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Contents Alexandria Journal of Hepatogastroenterology Volume IIIX ( II ) August 2013
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Disclaimer The Publisher the Egyptian Society of
Hepatology Gastroenterology and Infectious Diseases in
Alexandria and Editors cannot be held responsible for errors
or any consequences arising from the use of information
contained in this journal the views and opinions expressed
do not necessarily reflect the those of the Publisher The
Egyptian Society of Hepatology Gastroenterology amp
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Original Article
Hemostatic Disorders in Egyptian Chronic Hepatitis C
Patients with Liver Cirrhosis Possible Mechanisms and
Relation to Portal Vein Thrombosis
Ali El Kady1 Magdi El Bordini2 Ayman El Shayeb1 Nihal El Habachi3 and Reem Sherif1
Tropical Medicine1 Clinical Pathology2 and Physiology
Departments3 Faculty of Medicine Alexandria University
-------------------------------------------
Original Article
Last Minute Donor Exclusion in Living Donor Liver
Transplantation Impact on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1
Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1 1Department of HBP Surgery National Liver Istitute Egypt
2Department of Hepatology National Liver Institute Egypt
3Department of Anaesthesia National Liver Institute Egypt
-------------------------------------------
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor
Alpha Gene Polymorphisms on Therapeutic Response in
Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A
Elwazzan D
Department of Tropical Medicine Department of Clinical
Pathologyy Alexandria UniversityEgypt
-------------------------------------------
2
12
23
Original Article
Hemostatic Disorders in Egyptian Chronic Hepatitis C Patients with Liver
Cirrhosis Possible Mechanisms and Relation to Portal Vein Thrombosis
Ali El Kady1 Magdi El Bordini2 Ayman El Shayeb1 Nihal El Habachi3 and Reem Sherif1
Tropical Medicine1 Clinical Pathology2 and Physiology Departments3 Faculty of Medicine Alexandria University
ABSTRACT
Liver cirrhosis is frequently accompanied by complex alterations in the hemostatic system Von Willebrand factor
(vWF) which is considered as a marker of endothelial cell activation plays an essential role in hemostasis ADAMTS13
(a distintegrin- like and metalloproteinase with thrombospondin type 1 motifs 13) which is produced exclusively in
hepatic stellate cells (HSCs) cleaves multimeric vWF Although the interaction between ADAMTS 13 and vWF could
be considered as initial step in hemostasis little information has been available on this interaction in Egyptian cirrhotic
hepatitis C virus (HCV) patients Aim of the work this study aimed at exploration of the possible relationship
between plasma level of Von Willebrand factor (vWF) and ADAMTS13 implicated in hemostatic disorders in Egyptian
cirrhotic HCV patients and its relation to portal vein thrombosis Methods The study was conducted on 40 cirrhotic
HCV patients who were further subdivided according to Child Pugh classification Moreover 20 healthy subjects were
enrolled as controls Plasma vWF and ADAMTS 13 were measured by ELISA Results Mean plasma vWF was
significantly higher in patients with liver cirrhosis than in controls (P lt 005) In patients plasma vWF was significantly
higher in those with Child C than those with Child B and A On the other hand plasma ADAMTS 13 was significantly
lower in patients than controls In patients with liver cirrhosis plasma ADAMTS 13 was significantly lower in Child C
than Child B and A ones Furthermore a significant negative correlation was observed between ADAMTS 13 and vWF
in the studied group (P lt 005)Cirrhotic patients with portal vein thrombosis had significantly lower level of
ADAMTS 13 and higher level of vWF than controls (plt 005) Conclusion Egyptian cirrhotic chronic HCV patients
may experience a hemostatic disorders which are evidenced by high levels of vWF and concomitant low levels of
ADAMTS 13 suggesting the possible role of both endothelial and liver dysfunction in those patients Moreover
further studies are needed to explore different therapeutic areas aiming at elevating levels of ADAMTS 13 particularly
in those patients with portal vein thrombosis
Introduction
Hepatitis C virus (HCV) is estimated to infect
approximately 180 million people worldwide (1) However the prevalence of HCV infection
varies through out the world Egyptian
Demographic health survey (EDHS) in 2009
reported an overall anti-HCV antibody
prevalence of 147 and the number of
Egyptians estimated to be chronically infected
was 98 (2) Cirrhosis develops in
approximately 10 to 15 of individuals
with chronic HCV infection (3) There are
external and host factors such as older age at
time of infection male gender coinfection
with hepatitis B virus (HBV) and comorbid
conditions such as schistosomiasis that can
increase the risk of progression of liver
disease (4) Normal hemostasis results from
well regulated processes that maintain blood
in a fluid clot free state in normal vessels
while inducing rapid formation of a localized
plug at the site of vascular injury(5) The liver
plays a major role in hemostasis by
synthesizing all clotting factors (except von
Willebrand factor (vWF) coagulation
inhibitors and several fibrinolytic proteins
The hepatic reticuloendothelial system clears
activated clotting factors proteolytic enzyme
inhibitor complexes and fibrin degradation
products The normal hemostatic system
becomes severely impaired in cases of end
stage liver disease predisposing to bleeding (6) Intact blood vessels are the main
moderators of bloods tendency to clot The
endothelial cells of intact vessels prevent
blood clotting with the protein
thrombomodulin which facilitates anti-
coagulant and fibrinolytic activity by conv-
erting Protein C (PC) to activated Protein C
(APC)(7) Endothelial cells also prevent
platelet aggregation and promote vasodilation
with nitric oxide and prostacyclin Add-
itionally normal endothelium expresses other
anticoagulants such as glycosamino-glycans
and heparinoids and promotes fibrinolysis by
producing tissue plasminogen activator
(tPA)(8) In liver cirrhosis a sinusoidal
microcirculatory disturbance occurs when the
normal hepatic structure is disrupted by fibrin
deposition (9) or by impaired balance between
the action of vasoconstrictors and vasodilators
in hepatic vascular circulation (10) Various
classifications have been used to assess
severity of liver disease In patients with
chronic liver disease Childs grading with
Child Pughs classification modification is
frequently used It is useful in assessing
prognosis and estimating the potential risk of
variceal bleeding and operative mortality
This system uses a combination of clinical
and laboratory parameters (serum bilirubin
serum albumin prothrombin time ascites and
hepatic encephalopathy) The Child grade has
been equated with survival and Child C is the
most severe with an overall mortality of 88 (11) When endothelial injury occurs the end-
othelial cells stop secretion of coagulation and
aggregation inhibitors and instead secrete Von
Willebrand factor (vWF) (12) which is
considered as a marker of endothelial cell
activation (damage) and plays an essential
role in hemostasis(1314) vWF is a multimeric
large adhesive glycoprotein It mediates
platelet adhesion to exposed sub-endothelium
at sites of vascular injury under conditions of
stress Unusual large vWF multimers
(ULvWFMs) are produced exclusively in
vascular endothelial cells (ECS) and stored in
an intracellular organelle termed Weibel-
Palade bodies (WPBs) and released into the
circulation upon stimuli (15) The A1 domain
of VWF forms the principle binding sites for
platelet glycoprotein 1b (Gp1b) that promotes
platelet aggregation (16) ADAMTS13 (a
distintegrin- like and metalloproteinase with
thrombospondin type 1 motifs13) is a
metalloproteinase that specifically cleaves
multimeric vWF in the A2 domain (1314)
ADAMTS13 is produced exclusively in
hepatic stellate cells (HSCs)(17) Platelets (18)
vascular endothelial cells (19) and kidney
podocytes but the amount produced by these
cell types appears to be far less than that
produced by HSCs On the occurrence of liver
injury accompanied by a necroinflammatory
process the sinusoidal endothelial cells
become positive for vWF in association with
the capillarization of hepatic sinusoids(9) Sub-
sequently platelets adhere to subendothelial
tissue mediated by ULvWFMs ADAMTS13
then cleaves ULvWFMs into smaller vWF
multimers This interaction of ADAMTS13
and ULvWFMs is indeed the initial step in
hemostasis (1314) In the absence of ADAMTS
13 activity ULvWFMs are released from
vascular endothelial cells and improperly
cleaved causing them to accumulate and to
induce the formation of microvascular platelet
thrombi in the microvasculature under
conditions of high shear stress (14) However
little information has been available on the
interaction between vWF and ADAMTS 13 in
liver cirrhosis in Egyptian patients
Aim of Work
Our study aimed to explore the possible
relationship between plasma level of Von
Willebrand factor (vWF) and ADAMTS 13
implicated in hemostatic disorders in Egypt-
ian cirrhotic HCV patients and occurrence of
portal vein thrombosis
Subjects amp Methods
The protocol of the study was approved by
the Ethics committee of the Faculty of
Medicine University of Alexandria The
study was carried out on 60 patients classified
into two groups Group I included 40 patients
with chronic HCV with liver cirrhosis who
were subdivided according to Child- Pugh
classification (11) into three subgroups Group
Ia 10 patients with Child class A Group Ib
12 patients with Child class B and Group Ic
18 patients with Child class C Furthermore
20 healthy individuals with matching age and
sex were taken as controls (Group II) Patients
with history of alcohol abuse heart disease
kidney disease diabetes mellitus HBV
infection or malignancy were excluded from
the study All patients within group I were
HCV positive (HCV Ab +ve and confirmed
by PCR for HCV-RNA) All patients and
controls were interviewed and subjected to
the following 1Clinical evaluation and ultra-
sound examination for evidence of cirrhosis
2Routine laboratory investigations including
Complete blood picture Liver functions tests
(serum ALT AST GGT Alkaline phos-
phatase serum bilirubin serum albumin
prothrombin time and activity)(20) 3 Viral
markers for hepatitis B (HBs Ag) and
hepatitis C (Anti HCV) by ELISA(21) 4
Abdominal imaging using Ultrasound for
evidence of portal vein thrombosis
5Estimation of plasma ADAMTS13 levels
Estimation of plasma ADAMTS 13 activity
level was done using the Technozym
ADAMTS 13 ELISA test (22) 6- Plasma level
of von Willebrand factor using (vWF Ag
ELISA kit Technoclone Vienna Austria) (23)
Statistical Analysis
Data were collected revised and transferred
into the Statistical Package for social science
(SPSS version 10) Results were expressed
as means and standard deviation Statistical
tests used in this study were student t-test F-
test and Pearson correlation A level of 5
was considered as the cut off level of
significance
Results
The mean levels of plasma vWF was
significantly higher in patients compared to
control group (20012plusmn9544 and 7840plusmn331
μmoll respectively t= 523 p = 0001) (Table
I) In patients with liver cirrhosis (Gp I) the
mean value of plasma vWF was significantly
higher in Child C patients than Child B and A
ones (2697plusmn9633 17050plusmn1140 and 8530
plusmn4415 μmoll respectively) (F= 25 p= 000)
Moreover it was significantly higher in Child
B patients than A (Fig 1) On the other hand
the mean Plasma ADAMTS 13 activity was
significantly lower in patients than controls
2930plusmn1744 and 6540plusmn2303 respectively
(t= 677 p= 000)(Table I) Furthermore in
group I patients the mean values of
ADAMTS 13 activity were significantly
lower in Child C patients than Child B and A
ones (1716plusmn806 2558plusmn1111 and 5480
plusmn590 respectively) (F= 60 p= 000) and in
child B than in Child A patients (Fig 2)
Table I Mean plasma vWF and ADAMTS 13 in patients and controls
vWF(μmoll) ADAMTS 13 activity ()
Patients(n=40) 20012plusmn9544 2930plusmn 1744
Controls (n=20) 7840plusmn331 6540plusmn 2303
T 532 677
P 0001 0000
In group I 10 patients had portal vein
thrombosis when evaluated by ultrasound
examination ( 8 of them were Child C while 2
were Child B) however the remaining 30
patients had not The mean value of plasma
vWF was significantly higher in those with
portal vein thrombosis than those without it
(28840plusmn 10527 and 17070plusmn 7231 μmoll
respectively)(t= 393 P=0002) On the other
hand in the same group I the mean value of
ADAMTS 13 activity was significantly lower
in those with portal vein thrombosis than in
those without (1650plusmn 1000 and 335plusmn
1744 respectively) (t= 292 p= 0001)
(Table II)
Table II Mean plasma vWF and ADAMTS 13 in patients with and without PV thrombosis
vWF(μmoll) ADAMTS 13 activity ( )
PV Thrombosis (n=10) 28840plusmn 10527 1650plusmn 1000
No PV Thrombosis (n=30) 17070plusmn 7231 335plusmn 1744
T 393 292
P 0002 0001
Correlation studies (Table III) revealed that
significant positive correlation was found
between vWF and Child Pugh score ( r= 072
p= 000) (Fig 3) On the other hand
significant negative correlation was noticed
between ADAMTS 13 activity and each of
vWF and Child Pugh score in patients with
liver cirrhosis (GpI) ( r= - 051 and ndash 077 p=
000 0001) (Fig 4)
Table III correlation between ADAMTS 13 vWF and Child Pugh Score
ADAMTS 13 Child Pugh Score
vWF r= -051
p= 000
r= 072
p= 000
ADAMTS 13 r= -077
p= 0001
Figure 1 Mean Plasma vWF (umoll) in patients Figure 2 Mean Plasma ADAMTS 13 activity in patients
Child Score
141210864
vW
F
600
500
400
300
200
100
0
Figure 3 Correlation between vWF and Child Pugh score in patients
Figure 5 correlation between ADAMTS 13 and Child Pugh Score in patients
Adamts 13
706050403020100
Ch
ild S
co
re
14
12
10
8
6
4
Figure 4 Correlation between ADAMTS 13 and Child Pugh score in patients
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
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45 Tsai HM Lian EC Antibodies to von Willebrand
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339(22) 1585-94
46 Kume Y Ikeda H Inoue M Tejima K Tomiya T
et al Hepatic stellate cell damage may lead to
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48 Claus RA Bockmeyer CL Sossdorf M Losche
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49 Reiter RA Varadi K Turecek PL Jilma B Knobl
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50 Ishikawa M Uemura M Matsuyama T
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52 Wanless IR Wong F Blendis LM Greig P
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53 Pluta A Gutkowski K Hartleb M Coagulopathy
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54 Banno F Kokame K Okuda T Honda S Miyata S
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Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Original Article
Hemostatic Disorders in Egyptian Chronic Hepatitis C Patients with Liver
Cirrhosis Possible Mechanisms and Relation to Portal Vein Thrombosis
Ali El Kady1 Magdi El Bordini2 Ayman El Shayeb1 Nihal El Habachi3 and Reem Sherif1
Tropical Medicine1 Clinical Pathology2 and Physiology Departments3 Faculty of Medicine Alexandria University
ABSTRACT
Liver cirrhosis is frequently accompanied by complex alterations in the hemostatic system Von Willebrand factor
(vWF) which is considered as a marker of endothelial cell activation plays an essential role in hemostasis ADAMTS13
(a distintegrin- like and metalloproteinase with thrombospondin type 1 motifs 13) which is produced exclusively in
hepatic stellate cells (HSCs) cleaves multimeric vWF Although the interaction between ADAMTS 13 and vWF could
be considered as initial step in hemostasis little information has been available on this interaction in Egyptian cirrhotic
hepatitis C virus (HCV) patients Aim of the work this study aimed at exploration of the possible relationship
between plasma level of Von Willebrand factor (vWF) and ADAMTS13 implicated in hemostatic disorders in Egyptian
cirrhotic HCV patients and its relation to portal vein thrombosis Methods The study was conducted on 40 cirrhotic
HCV patients who were further subdivided according to Child Pugh classification Moreover 20 healthy subjects were
enrolled as controls Plasma vWF and ADAMTS 13 were measured by ELISA Results Mean plasma vWF was
significantly higher in patients with liver cirrhosis than in controls (P lt 005) In patients plasma vWF was significantly
higher in those with Child C than those with Child B and A On the other hand plasma ADAMTS 13 was significantly
lower in patients than controls In patients with liver cirrhosis plasma ADAMTS 13 was significantly lower in Child C
than Child B and A ones Furthermore a significant negative correlation was observed between ADAMTS 13 and vWF
in the studied group (P lt 005)Cirrhotic patients with portal vein thrombosis had significantly lower level of
ADAMTS 13 and higher level of vWF than controls (plt 005) Conclusion Egyptian cirrhotic chronic HCV patients
may experience a hemostatic disorders which are evidenced by high levels of vWF and concomitant low levels of
ADAMTS 13 suggesting the possible role of both endothelial and liver dysfunction in those patients Moreover
further studies are needed to explore different therapeutic areas aiming at elevating levels of ADAMTS 13 particularly
in those patients with portal vein thrombosis
Introduction
Hepatitis C virus (HCV) is estimated to infect
approximately 180 million people worldwide (1) However the prevalence of HCV infection
varies through out the world Egyptian
Demographic health survey (EDHS) in 2009
reported an overall anti-HCV antibody
prevalence of 147 and the number of
Egyptians estimated to be chronically infected
was 98 (2) Cirrhosis develops in
approximately 10 to 15 of individuals
with chronic HCV infection (3) There are
external and host factors such as older age at
time of infection male gender coinfection
with hepatitis B virus (HBV) and comorbid
conditions such as schistosomiasis that can
increase the risk of progression of liver
disease (4) Normal hemostasis results from
well regulated processes that maintain blood
in a fluid clot free state in normal vessels
while inducing rapid formation of a localized
plug at the site of vascular injury(5) The liver
plays a major role in hemostasis by
synthesizing all clotting factors (except von
Willebrand factor (vWF) coagulation
inhibitors and several fibrinolytic proteins
The hepatic reticuloendothelial system clears
activated clotting factors proteolytic enzyme
inhibitor complexes and fibrin degradation
products The normal hemostatic system
becomes severely impaired in cases of end
stage liver disease predisposing to bleeding (6) Intact blood vessels are the main
moderators of bloods tendency to clot The
endothelial cells of intact vessels prevent
blood clotting with the protein
thrombomodulin which facilitates anti-
coagulant and fibrinolytic activity by conv-
erting Protein C (PC) to activated Protein C
(APC)(7) Endothelial cells also prevent
platelet aggregation and promote vasodilation
with nitric oxide and prostacyclin Add-
itionally normal endothelium expresses other
anticoagulants such as glycosamino-glycans
and heparinoids and promotes fibrinolysis by
producing tissue plasminogen activator
(tPA)(8) In liver cirrhosis a sinusoidal
microcirculatory disturbance occurs when the
normal hepatic structure is disrupted by fibrin
deposition (9) or by impaired balance between
the action of vasoconstrictors and vasodilators
in hepatic vascular circulation (10) Various
classifications have been used to assess
severity of liver disease In patients with
chronic liver disease Childs grading with
Child Pughs classification modification is
frequently used It is useful in assessing
prognosis and estimating the potential risk of
variceal bleeding and operative mortality
This system uses a combination of clinical
and laboratory parameters (serum bilirubin
serum albumin prothrombin time ascites and
hepatic encephalopathy) The Child grade has
been equated with survival and Child C is the
most severe with an overall mortality of 88 (11) When endothelial injury occurs the end-
othelial cells stop secretion of coagulation and
aggregation inhibitors and instead secrete Von
Willebrand factor (vWF) (12) which is
considered as a marker of endothelial cell
activation (damage) and plays an essential
role in hemostasis(1314) vWF is a multimeric
large adhesive glycoprotein It mediates
platelet adhesion to exposed sub-endothelium
at sites of vascular injury under conditions of
stress Unusual large vWF multimers
(ULvWFMs) are produced exclusively in
vascular endothelial cells (ECS) and stored in
an intracellular organelle termed Weibel-
Palade bodies (WPBs) and released into the
circulation upon stimuli (15) The A1 domain
of VWF forms the principle binding sites for
platelet glycoprotein 1b (Gp1b) that promotes
platelet aggregation (16) ADAMTS13 (a
distintegrin- like and metalloproteinase with
thrombospondin type 1 motifs13) is a
metalloproteinase that specifically cleaves
multimeric vWF in the A2 domain (1314)
ADAMTS13 is produced exclusively in
hepatic stellate cells (HSCs)(17) Platelets (18)
vascular endothelial cells (19) and kidney
podocytes but the amount produced by these
cell types appears to be far less than that
produced by HSCs On the occurrence of liver
injury accompanied by a necroinflammatory
process the sinusoidal endothelial cells
become positive for vWF in association with
the capillarization of hepatic sinusoids(9) Sub-
sequently platelets adhere to subendothelial
tissue mediated by ULvWFMs ADAMTS13
then cleaves ULvWFMs into smaller vWF
multimers This interaction of ADAMTS13
and ULvWFMs is indeed the initial step in
hemostasis (1314) In the absence of ADAMTS
13 activity ULvWFMs are released from
vascular endothelial cells and improperly
cleaved causing them to accumulate and to
induce the formation of microvascular platelet
thrombi in the microvasculature under
conditions of high shear stress (14) However
little information has been available on the
interaction between vWF and ADAMTS 13 in
liver cirrhosis in Egyptian patients
Aim of Work
Our study aimed to explore the possible
relationship between plasma level of Von
Willebrand factor (vWF) and ADAMTS 13
implicated in hemostatic disorders in Egypt-
ian cirrhotic HCV patients and occurrence of
portal vein thrombosis
Subjects amp Methods
The protocol of the study was approved by
the Ethics committee of the Faculty of
Medicine University of Alexandria The
study was carried out on 60 patients classified
into two groups Group I included 40 patients
with chronic HCV with liver cirrhosis who
were subdivided according to Child- Pugh
classification (11) into three subgroups Group
Ia 10 patients with Child class A Group Ib
12 patients with Child class B and Group Ic
18 patients with Child class C Furthermore
20 healthy individuals with matching age and
sex were taken as controls (Group II) Patients
with history of alcohol abuse heart disease
kidney disease diabetes mellitus HBV
infection or malignancy were excluded from
the study All patients within group I were
HCV positive (HCV Ab +ve and confirmed
by PCR for HCV-RNA) All patients and
controls were interviewed and subjected to
the following 1Clinical evaluation and ultra-
sound examination for evidence of cirrhosis
2Routine laboratory investigations including
Complete blood picture Liver functions tests
(serum ALT AST GGT Alkaline phos-
phatase serum bilirubin serum albumin
prothrombin time and activity)(20) 3 Viral
markers for hepatitis B (HBs Ag) and
hepatitis C (Anti HCV) by ELISA(21) 4
Abdominal imaging using Ultrasound for
evidence of portal vein thrombosis
5Estimation of plasma ADAMTS13 levels
Estimation of plasma ADAMTS 13 activity
level was done using the Technozym
ADAMTS 13 ELISA test (22) 6- Plasma level
of von Willebrand factor using (vWF Ag
ELISA kit Technoclone Vienna Austria) (23)
Statistical Analysis
Data were collected revised and transferred
into the Statistical Package for social science
(SPSS version 10) Results were expressed
as means and standard deviation Statistical
tests used in this study were student t-test F-
test and Pearson correlation A level of 5
was considered as the cut off level of
significance
Results
The mean levels of plasma vWF was
significantly higher in patients compared to
control group (20012plusmn9544 and 7840plusmn331
μmoll respectively t= 523 p = 0001) (Table
I) In patients with liver cirrhosis (Gp I) the
mean value of plasma vWF was significantly
higher in Child C patients than Child B and A
ones (2697plusmn9633 17050plusmn1140 and 8530
plusmn4415 μmoll respectively) (F= 25 p= 000)
Moreover it was significantly higher in Child
B patients than A (Fig 1) On the other hand
the mean Plasma ADAMTS 13 activity was
significantly lower in patients than controls
2930plusmn1744 and 6540plusmn2303 respectively
(t= 677 p= 000)(Table I) Furthermore in
group I patients the mean values of
ADAMTS 13 activity were significantly
lower in Child C patients than Child B and A
ones (1716plusmn806 2558plusmn1111 and 5480
plusmn590 respectively) (F= 60 p= 000) and in
child B than in Child A patients (Fig 2)
Table I Mean plasma vWF and ADAMTS 13 in patients and controls
vWF(μmoll) ADAMTS 13 activity ()
Patients(n=40) 20012plusmn9544 2930plusmn 1744
Controls (n=20) 7840plusmn331 6540plusmn 2303
T 532 677
P 0001 0000
In group I 10 patients had portal vein
thrombosis when evaluated by ultrasound
examination ( 8 of them were Child C while 2
were Child B) however the remaining 30
patients had not The mean value of plasma
vWF was significantly higher in those with
portal vein thrombosis than those without it
(28840plusmn 10527 and 17070plusmn 7231 μmoll
respectively)(t= 393 P=0002) On the other
hand in the same group I the mean value of
ADAMTS 13 activity was significantly lower
in those with portal vein thrombosis than in
those without (1650plusmn 1000 and 335plusmn
1744 respectively) (t= 292 p= 0001)
(Table II)
Table II Mean plasma vWF and ADAMTS 13 in patients with and without PV thrombosis
vWF(μmoll) ADAMTS 13 activity ( )
PV Thrombosis (n=10) 28840plusmn 10527 1650plusmn 1000
No PV Thrombosis (n=30) 17070plusmn 7231 335plusmn 1744
T 393 292
P 0002 0001
Correlation studies (Table III) revealed that
significant positive correlation was found
between vWF and Child Pugh score ( r= 072
p= 000) (Fig 3) On the other hand
significant negative correlation was noticed
between ADAMTS 13 activity and each of
vWF and Child Pugh score in patients with
liver cirrhosis (GpI) ( r= - 051 and ndash 077 p=
000 0001) (Fig 4)
Table III correlation between ADAMTS 13 vWF and Child Pugh Score
ADAMTS 13 Child Pugh Score
vWF r= -051
p= 000
r= 072
p= 000
ADAMTS 13 r= -077
p= 0001
Figure 1 Mean Plasma vWF (umoll) in patients Figure 2 Mean Plasma ADAMTS 13 activity in patients
Child Score
141210864
vW
F
600
500
400
300
200
100
0
Figure 3 Correlation between vWF and Child Pugh score in patients
Figure 5 correlation between ADAMTS 13 and Child Pugh Score in patients
Adamts 13
706050403020100
Ch
ild S
co
re
14
12
10
8
6
4
Figure 4 Correlation between ADAMTS 13 and Child Pugh score in patients
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
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enhanced cytokinemia and plasma inhibitor on the
decreased activity of plasma ADAMTS13 in
patients with alcoholic hepatitis relationship to
endotoxemia Alcoholism Clinical and
Experimental Research 2010 34(1) 25-33
51 Amitrano L Guardascione MA Brancaccio V
Margaglione M Manguso F et al Risk factors and
clinical presentation of portal vein thrombosis in
patients with liver cirrhosis Journal of Hepatology
2004 40(5) 736-41
52 Wanless IR Wong F Blendis LM Greig P
Heathcote EJ et al Hepatic and portal vein
thrombosis in cirrhosis possible role in
development of parenchymal extinction and portal
hypertension Hepatology 1995 21(5) 1238-47
53 Pluta A Gutkowski K Hartleb M Coagulopathy
in liver diseases Advanced Medical Science 2010
55 16-21
54 Banno F Kokame K Okuda T Honda S Miyata S
et al Complete deficiency in ADAMTS13 is
prothrombotic but alone is not sufficient to cause
thrombotic thrombocytopenic purpura Blood
2006 107 3161-6
Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
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Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
products The normal hemostatic system
becomes severely impaired in cases of end
stage liver disease predisposing to bleeding (6) Intact blood vessels are the main
moderators of bloods tendency to clot The
endothelial cells of intact vessels prevent
blood clotting with the protein
thrombomodulin which facilitates anti-
coagulant and fibrinolytic activity by conv-
erting Protein C (PC) to activated Protein C
(APC)(7) Endothelial cells also prevent
platelet aggregation and promote vasodilation
with nitric oxide and prostacyclin Add-
itionally normal endothelium expresses other
anticoagulants such as glycosamino-glycans
and heparinoids and promotes fibrinolysis by
producing tissue plasminogen activator
(tPA)(8) In liver cirrhosis a sinusoidal
microcirculatory disturbance occurs when the
normal hepatic structure is disrupted by fibrin
deposition (9) or by impaired balance between
the action of vasoconstrictors and vasodilators
in hepatic vascular circulation (10) Various
classifications have been used to assess
severity of liver disease In patients with
chronic liver disease Childs grading with
Child Pughs classification modification is
frequently used It is useful in assessing
prognosis and estimating the potential risk of
variceal bleeding and operative mortality
This system uses a combination of clinical
and laboratory parameters (serum bilirubin
serum albumin prothrombin time ascites and
hepatic encephalopathy) The Child grade has
been equated with survival and Child C is the
most severe with an overall mortality of 88 (11) When endothelial injury occurs the end-
othelial cells stop secretion of coagulation and
aggregation inhibitors and instead secrete Von
Willebrand factor (vWF) (12) which is
considered as a marker of endothelial cell
activation (damage) and plays an essential
role in hemostasis(1314) vWF is a multimeric
large adhesive glycoprotein It mediates
platelet adhesion to exposed sub-endothelium
at sites of vascular injury under conditions of
stress Unusual large vWF multimers
(ULvWFMs) are produced exclusively in
vascular endothelial cells (ECS) and stored in
an intracellular organelle termed Weibel-
Palade bodies (WPBs) and released into the
circulation upon stimuli (15) The A1 domain
of VWF forms the principle binding sites for
platelet glycoprotein 1b (Gp1b) that promotes
platelet aggregation (16) ADAMTS13 (a
distintegrin- like and metalloproteinase with
thrombospondin type 1 motifs13) is a
metalloproteinase that specifically cleaves
multimeric vWF in the A2 domain (1314)
ADAMTS13 is produced exclusively in
hepatic stellate cells (HSCs)(17) Platelets (18)
vascular endothelial cells (19) and kidney
podocytes but the amount produced by these
cell types appears to be far less than that
produced by HSCs On the occurrence of liver
injury accompanied by a necroinflammatory
process the sinusoidal endothelial cells
become positive for vWF in association with
the capillarization of hepatic sinusoids(9) Sub-
sequently platelets adhere to subendothelial
tissue mediated by ULvWFMs ADAMTS13
then cleaves ULvWFMs into smaller vWF
multimers This interaction of ADAMTS13
and ULvWFMs is indeed the initial step in
hemostasis (1314) In the absence of ADAMTS
13 activity ULvWFMs are released from
vascular endothelial cells and improperly
cleaved causing them to accumulate and to
induce the formation of microvascular platelet
thrombi in the microvasculature under
conditions of high shear stress (14) However
little information has been available on the
interaction between vWF and ADAMTS 13 in
liver cirrhosis in Egyptian patients
Aim of Work
Our study aimed to explore the possible
relationship between plasma level of Von
Willebrand factor (vWF) and ADAMTS 13
implicated in hemostatic disorders in Egypt-
ian cirrhotic HCV patients and occurrence of
portal vein thrombosis
Subjects amp Methods
The protocol of the study was approved by
the Ethics committee of the Faculty of
Medicine University of Alexandria The
study was carried out on 60 patients classified
into two groups Group I included 40 patients
with chronic HCV with liver cirrhosis who
were subdivided according to Child- Pugh
classification (11) into three subgroups Group
Ia 10 patients with Child class A Group Ib
12 patients with Child class B and Group Ic
18 patients with Child class C Furthermore
20 healthy individuals with matching age and
sex were taken as controls (Group II) Patients
with history of alcohol abuse heart disease
kidney disease diabetes mellitus HBV
infection or malignancy were excluded from
the study All patients within group I were
HCV positive (HCV Ab +ve and confirmed
by PCR for HCV-RNA) All patients and
controls were interviewed and subjected to
the following 1Clinical evaluation and ultra-
sound examination for evidence of cirrhosis
2Routine laboratory investigations including
Complete blood picture Liver functions tests
(serum ALT AST GGT Alkaline phos-
phatase serum bilirubin serum albumin
prothrombin time and activity)(20) 3 Viral
markers for hepatitis B (HBs Ag) and
hepatitis C (Anti HCV) by ELISA(21) 4
Abdominal imaging using Ultrasound for
evidence of portal vein thrombosis
5Estimation of plasma ADAMTS13 levels
Estimation of plasma ADAMTS 13 activity
level was done using the Technozym
ADAMTS 13 ELISA test (22) 6- Plasma level
of von Willebrand factor using (vWF Ag
ELISA kit Technoclone Vienna Austria) (23)
Statistical Analysis
Data were collected revised and transferred
into the Statistical Package for social science
(SPSS version 10) Results were expressed
as means and standard deviation Statistical
tests used in this study were student t-test F-
test and Pearson correlation A level of 5
was considered as the cut off level of
significance
Results
The mean levels of plasma vWF was
significantly higher in patients compared to
control group (20012plusmn9544 and 7840plusmn331
μmoll respectively t= 523 p = 0001) (Table
I) In patients with liver cirrhosis (Gp I) the
mean value of plasma vWF was significantly
higher in Child C patients than Child B and A
ones (2697plusmn9633 17050plusmn1140 and 8530
plusmn4415 μmoll respectively) (F= 25 p= 000)
Moreover it was significantly higher in Child
B patients than A (Fig 1) On the other hand
the mean Plasma ADAMTS 13 activity was
significantly lower in patients than controls
2930plusmn1744 and 6540plusmn2303 respectively
(t= 677 p= 000)(Table I) Furthermore in
group I patients the mean values of
ADAMTS 13 activity were significantly
lower in Child C patients than Child B and A
ones (1716plusmn806 2558plusmn1111 and 5480
plusmn590 respectively) (F= 60 p= 000) and in
child B than in Child A patients (Fig 2)
Table I Mean plasma vWF and ADAMTS 13 in patients and controls
vWF(μmoll) ADAMTS 13 activity ()
Patients(n=40) 20012plusmn9544 2930plusmn 1744
Controls (n=20) 7840plusmn331 6540plusmn 2303
T 532 677
P 0001 0000
In group I 10 patients had portal vein
thrombosis when evaluated by ultrasound
examination ( 8 of them were Child C while 2
were Child B) however the remaining 30
patients had not The mean value of plasma
vWF was significantly higher in those with
portal vein thrombosis than those without it
(28840plusmn 10527 and 17070plusmn 7231 μmoll
respectively)(t= 393 P=0002) On the other
hand in the same group I the mean value of
ADAMTS 13 activity was significantly lower
in those with portal vein thrombosis than in
those without (1650plusmn 1000 and 335plusmn
1744 respectively) (t= 292 p= 0001)
(Table II)
Table II Mean plasma vWF and ADAMTS 13 in patients with and without PV thrombosis
vWF(μmoll) ADAMTS 13 activity ( )
PV Thrombosis (n=10) 28840plusmn 10527 1650plusmn 1000
No PV Thrombosis (n=30) 17070plusmn 7231 335plusmn 1744
T 393 292
P 0002 0001
Correlation studies (Table III) revealed that
significant positive correlation was found
between vWF and Child Pugh score ( r= 072
p= 000) (Fig 3) On the other hand
significant negative correlation was noticed
between ADAMTS 13 activity and each of
vWF and Child Pugh score in patients with
liver cirrhosis (GpI) ( r= - 051 and ndash 077 p=
000 0001) (Fig 4)
Table III correlation between ADAMTS 13 vWF and Child Pugh Score
ADAMTS 13 Child Pugh Score
vWF r= -051
p= 000
r= 072
p= 000
ADAMTS 13 r= -077
p= 0001
Figure 1 Mean Plasma vWF (umoll) in patients Figure 2 Mean Plasma ADAMTS 13 activity in patients
Child Score
141210864
vW
F
600
500
400
300
200
100
0
Figure 3 Correlation between vWF and Child Pugh score in patients
Figure 5 correlation between ADAMTS 13 and Child Pugh Score in patients
Adamts 13
706050403020100
Ch
ild S
co
re
14
12
10
8
6
4
Figure 4 Correlation between ADAMTS 13 and Child Pugh score in patients
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
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38 Uemura M Fujimura Y Matsumoto M Ishizashi
H Kato S et al Comprehensive analysis of
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29
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Elevated levels of von Willebrand factor in
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JF Effects of inflammatory cytokines on the
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ultralarge von Willebrand factor multimers under
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43 Cao WJ Niiya M Zheng XW Shang DZ Zheng
XL Inflammatory cytokines inhibit ADAMTS13
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Med 1998 339(22) 1578-84
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339(22) 1585-94
46 Kume Y Ikeda H Inoue M Tejima K Tomiya T
et al Hepatic stellate cell damage may lead to
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Dockal M et al Increased ADAMTS-13
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W The balance between von-Willebrand factor
and its cleaving protease ADAMTS13 biomarker
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10(2) 236-48
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P Changes in ADAMTS13 (von-Willebrand-
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Haemostasis 2005 93(3) 554-8
50 Ishikawa M Uemura M Matsuyama T
Matsumoto M Ishizashi H et al Potential role of
enhanced cytokinemia and plasma inhibitor on the
decreased activity of plasma ADAMTS13 in
patients with alcoholic hepatitis relationship to
endotoxemia Alcoholism Clinical and
Experimental Research 2010 34(1) 25-33
51 Amitrano L Guardascione MA Brancaccio V
Margaglione M Manguso F et al Risk factors and
clinical presentation of portal vein thrombosis in
patients with liver cirrhosis Journal of Hepatology
2004 40(5) 736-41
52 Wanless IR Wong F Blendis LM Greig P
Heathcote EJ et al Hepatic and portal vein
thrombosis in cirrhosis possible role in
development of parenchymal extinction and portal
hypertension Hepatology 1995 21(5) 1238-47
53 Pluta A Gutkowski K Hartleb M Coagulopathy
in liver diseases Advanced Medical Science 2010
55 16-21
54 Banno F Kokame K Okuda T Honda S Miyata S
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Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Aim of Work
Our study aimed to explore the possible
relationship between plasma level of Von
Willebrand factor (vWF) and ADAMTS 13
implicated in hemostatic disorders in Egypt-
ian cirrhotic HCV patients and occurrence of
portal vein thrombosis
Subjects amp Methods
The protocol of the study was approved by
the Ethics committee of the Faculty of
Medicine University of Alexandria The
study was carried out on 60 patients classified
into two groups Group I included 40 patients
with chronic HCV with liver cirrhosis who
were subdivided according to Child- Pugh
classification (11) into three subgroups Group
Ia 10 patients with Child class A Group Ib
12 patients with Child class B and Group Ic
18 patients with Child class C Furthermore
20 healthy individuals with matching age and
sex were taken as controls (Group II) Patients
with history of alcohol abuse heart disease
kidney disease diabetes mellitus HBV
infection or malignancy were excluded from
the study All patients within group I were
HCV positive (HCV Ab +ve and confirmed
by PCR for HCV-RNA) All patients and
controls were interviewed and subjected to
the following 1Clinical evaluation and ultra-
sound examination for evidence of cirrhosis
2Routine laboratory investigations including
Complete blood picture Liver functions tests
(serum ALT AST GGT Alkaline phos-
phatase serum bilirubin serum albumin
prothrombin time and activity)(20) 3 Viral
markers for hepatitis B (HBs Ag) and
hepatitis C (Anti HCV) by ELISA(21) 4
Abdominal imaging using Ultrasound for
evidence of portal vein thrombosis
5Estimation of plasma ADAMTS13 levels
Estimation of plasma ADAMTS 13 activity
level was done using the Technozym
ADAMTS 13 ELISA test (22) 6- Plasma level
of von Willebrand factor using (vWF Ag
ELISA kit Technoclone Vienna Austria) (23)
Statistical Analysis
Data were collected revised and transferred
into the Statistical Package for social science
(SPSS version 10) Results were expressed
as means and standard deviation Statistical
tests used in this study were student t-test F-
test and Pearson correlation A level of 5
was considered as the cut off level of
significance
Results
The mean levels of plasma vWF was
significantly higher in patients compared to
control group (20012plusmn9544 and 7840plusmn331
μmoll respectively t= 523 p = 0001) (Table
I) In patients with liver cirrhosis (Gp I) the
mean value of plasma vWF was significantly
higher in Child C patients than Child B and A
ones (2697plusmn9633 17050plusmn1140 and 8530
plusmn4415 μmoll respectively) (F= 25 p= 000)
Moreover it was significantly higher in Child
B patients than A (Fig 1) On the other hand
the mean Plasma ADAMTS 13 activity was
significantly lower in patients than controls
2930plusmn1744 and 6540plusmn2303 respectively
(t= 677 p= 000)(Table I) Furthermore in
group I patients the mean values of
ADAMTS 13 activity were significantly
lower in Child C patients than Child B and A
ones (1716plusmn806 2558plusmn1111 and 5480
plusmn590 respectively) (F= 60 p= 000) and in
child B than in Child A patients (Fig 2)
Table I Mean plasma vWF and ADAMTS 13 in patients and controls
vWF(μmoll) ADAMTS 13 activity ()
Patients(n=40) 20012plusmn9544 2930plusmn 1744
Controls (n=20) 7840plusmn331 6540plusmn 2303
T 532 677
P 0001 0000
In group I 10 patients had portal vein
thrombosis when evaluated by ultrasound
examination ( 8 of them were Child C while 2
were Child B) however the remaining 30
patients had not The mean value of plasma
vWF was significantly higher in those with
portal vein thrombosis than those without it
(28840plusmn 10527 and 17070plusmn 7231 μmoll
respectively)(t= 393 P=0002) On the other
hand in the same group I the mean value of
ADAMTS 13 activity was significantly lower
in those with portal vein thrombosis than in
those without (1650plusmn 1000 and 335plusmn
1744 respectively) (t= 292 p= 0001)
(Table II)
Table II Mean plasma vWF and ADAMTS 13 in patients with and without PV thrombosis
vWF(μmoll) ADAMTS 13 activity ( )
PV Thrombosis (n=10) 28840plusmn 10527 1650plusmn 1000
No PV Thrombosis (n=30) 17070plusmn 7231 335plusmn 1744
T 393 292
P 0002 0001
Correlation studies (Table III) revealed that
significant positive correlation was found
between vWF and Child Pugh score ( r= 072
p= 000) (Fig 3) On the other hand
significant negative correlation was noticed
between ADAMTS 13 activity and each of
vWF and Child Pugh score in patients with
liver cirrhosis (GpI) ( r= - 051 and ndash 077 p=
000 0001) (Fig 4)
Table III correlation between ADAMTS 13 vWF and Child Pugh Score
ADAMTS 13 Child Pugh Score
vWF r= -051
p= 000
r= 072
p= 000
ADAMTS 13 r= -077
p= 0001
Figure 1 Mean Plasma vWF (umoll) in patients Figure 2 Mean Plasma ADAMTS 13 activity in patients
Child Score
141210864
vW
F
600
500
400
300
200
100
0
Figure 3 Correlation between vWF and Child Pugh score in patients
Figure 5 correlation between ADAMTS 13 and Child Pugh Score in patients
Adamts 13
706050403020100
Ch
ild S
co
re
14
12
10
8
6
4
Figure 4 Correlation between ADAMTS 13 and Child Pugh score in patients
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
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JF Effects of inflammatory cytokines on the
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XL Inflammatory cytokines inhibit ADAMTS13
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339(22) 1585-94
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et al Hepatic stellate cell damage may lead to
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Dockal M et al Increased ADAMTS-13
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W The balance between von-Willebrand factor
and its cleaving protease ADAMTS13 biomarker
in systemic inflammation and development of
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10(2) 236-48
49 Reiter RA Varadi K Turecek PL Jilma B Knobl
P Changes in ADAMTS13 (von-Willebrand-
factorcleaving protease) activity after induced
release of von Willebrand factor during acute
systemic inflammation Thrombosis and
Haemostasis 2005 93(3) 554-8
50 Ishikawa M Uemura M Matsuyama T
Matsumoto M Ishizashi H et al Potential role of
enhanced cytokinemia and plasma inhibitor on the
decreased activity of plasma ADAMTS13 in
patients with alcoholic hepatitis relationship to
endotoxemia Alcoholism Clinical and
Experimental Research 2010 34(1) 25-33
51 Amitrano L Guardascione MA Brancaccio V
Margaglione M Manguso F et al Risk factors and
clinical presentation of portal vein thrombosis in
patients with liver cirrhosis Journal of Hepatology
2004 40(5) 736-41
52 Wanless IR Wong F Blendis LM Greig P
Heathcote EJ et al Hepatic and portal vein
thrombosis in cirrhosis possible role in
development of parenchymal extinction and portal
hypertension Hepatology 1995 21(5) 1238-47
53 Pluta A Gutkowski K Hartleb M Coagulopathy
in liver diseases Advanced Medical Science 2010
55 16-21
54 Banno F Kokame K Okuda T Honda S Miyata S
et al Complete deficiency in ADAMTS13 is
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2006 107 3161-6
Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Table I Mean plasma vWF and ADAMTS 13 in patients and controls
vWF(μmoll) ADAMTS 13 activity ()
Patients(n=40) 20012plusmn9544 2930plusmn 1744
Controls (n=20) 7840plusmn331 6540plusmn 2303
T 532 677
P 0001 0000
In group I 10 patients had portal vein
thrombosis when evaluated by ultrasound
examination ( 8 of them were Child C while 2
were Child B) however the remaining 30
patients had not The mean value of plasma
vWF was significantly higher in those with
portal vein thrombosis than those without it
(28840plusmn 10527 and 17070plusmn 7231 μmoll
respectively)(t= 393 P=0002) On the other
hand in the same group I the mean value of
ADAMTS 13 activity was significantly lower
in those with portal vein thrombosis than in
those without (1650plusmn 1000 and 335plusmn
1744 respectively) (t= 292 p= 0001)
(Table II)
Table II Mean plasma vWF and ADAMTS 13 in patients with and without PV thrombosis
vWF(μmoll) ADAMTS 13 activity ( )
PV Thrombosis (n=10) 28840plusmn 10527 1650plusmn 1000
No PV Thrombosis (n=30) 17070plusmn 7231 335plusmn 1744
T 393 292
P 0002 0001
Correlation studies (Table III) revealed that
significant positive correlation was found
between vWF and Child Pugh score ( r= 072
p= 000) (Fig 3) On the other hand
significant negative correlation was noticed
between ADAMTS 13 activity and each of
vWF and Child Pugh score in patients with
liver cirrhosis (GpI) ( r= - 051 and ndash 077 p=
000 0001) (Fig 4)
Table III correlation between ADAMTS 13 vWF and Child Pugh Score
ADAMTS 13 Child Pugh Score
vWF r= -051
p= 000
r= 072
p= 000
ADAMTS 13 r= -077
p= 0001
Figure 1 Mean Plasma vWF (umoll) in patients Figure 2 Mean Plasma ADAMTS 13 activity in patients
Child Score
141210864
vW
F
600
500
400
300
200
100
0
Figure 3 Correlation between vWF and Child Pugh score in patients
Figure 5 correlation between ADAMTS 13 and Child Pugh Score in patients
Adamts 13
706050403020100
Ch
ild S
co
re
14
12
10
8
6
4
Figure 4 Correlation between ADAMTS 13 and Child Pugh score in patients
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
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to the identification of the hepatitis C virus
Journal of Hepatology 2009 5 939-48
2 El Zanaty F Way A Egypt demographic and
health survey 2008 Cairo Egypt Ministry of
Health EL- Zanaty and associates and Macro
International 2009
3 Wilkins T Malcolm JK Raina D Hepatitis C
diagnosis and treatment American family
physician 2010 81(11) 1351-7
4 Ragni MV Belle SH Impact of human
immunodeficiency virus infection on progression
to end-stage liver disease in individuals with
hemophilia and hepatitis C virus infection J Infect
Dis 2001 183(7) 1112-5
5 Kumar V Abbas AK Faousto N Robbins and
Cotran Pathologic basis of disease 2005 (7)
Philadelphia Elsevier
6 Lisman T Leebeek FW de Groot PG
Haemostatic abnormalities in patients with liver
disease J Hepatol 2002 37 280-7
7 Furie B Furie BC Thrombus formation in vivo J
Clin Invest 2005 115(12) 3355-62
8 Pallister CJ Watson MS Haematology Scion
Publishing 2010 2 334-6
9 Schaffner F Popper H Capillarization of hepatic
sinusoids in man Gastroenterology 1963 44 239-
42
10 Martell M Coll M Raurell I Ezkurdia N Raurell
I et al Pathophysiology of splanchnic
vasodilatation in portal hypertension World J
Hepatol 2010 2(6) 208-20
11 Albers I Hartman H Bircher J Superiority of the
Child Pugh classification to quantitative liver
function tests for assessing prognosis of liver
cirrhosis Scand J Gastroenterol 1989 24 269-76
12 Ruggeri ZM Ware J The structure and function of
von Willebrand factor Thromb Haemost 1992 67
594-8
13 Moake JL Thrombotic microangiopathies New
Engl J M 2002 347(8) 589-600
14 Fujimura Y Matsumoto M Yagi H Yoshioka A
Matsui T et al Von Willebrand factor-cleaving
protease and Upshaw-Schulman syndrome Int J of
Hematol 2002 75(1) 25-34
15 Padilla A Moake JL Bernardo A Ball C Wang Y
et al P-selectin anchors newly released ultralarge
von Willebrand factor multimers to the endothelial
cell surface Blood 2004 103(6) 2150-6
16 Emsley J Cruz M Handin R Liddington R
Crystal structure of the von Willebrand factor A1
domain and implication for the binding of platelet
glycoprotein 1b J Biol chem 1998 273 361-401
17 Uemura M Tatsumi K Matsumoto M Fujimoto
M Matsuyama T et al Localization of
ADAMTS13 to the stellate cells of human liver
Blood 2005 106(3) 922-24
18 Suzuki M Murata M Matsubara Y Von
Willebrand factor-cleaving protease (ADAMTS-
13) in human platelets Biochemical and
Biophysical Research Communications 2004
313(1) 212-16
19 Turner N Nolasco L Tao Z Dong JF Moak J
Human endothelial cells synthesize and release
ADAMTS- 13 Journal of Thrombosis and
Haemostasis 2006 4(6) 1396-404
20 Burits H Ashwood R Tietz fundamentals of
clinical chemistry 4th ed Philadelphia Saunders
WB 1996 82-5
21 Martin P Friedman S Dienstag L Diagnostic
approach to viral hepatitis In Zuckerman J
Thomas C Viral hepatitis Scientific basis and
clinical management UK Longman Group 1993
393- 409
22 Sadler JE A new name in thrombosis
ADAMTS13 Proc Natl Acad Sci U S A 2002 99
552-4
23 Deng L Bremme K Hansson LO Blomback M
Plasma levels of von Willebrand factor and
fibronectin as markers of persisting endothelial
damage in preeclampsia Obstet Gynecol 1994
84(6) 941-5
24 Violi F Ferro D Basili S Quintarelli C Musca A
et al Hyperfibrinolysis resulting from clotting
activation in patients with different stages of liver
cirrhosis Hepatology 1993 17 78-83
25 Adams DH Burra P Hubscher SG Elias E
Newman W Endothelial activation and circulating
vascular adhesion molecules in alcoholic liver
disease Hepatology 1994 19 588-94
26 Northup PG Sundaram V Fallon MB reddy KR
Balogun RA et al Hypercoagulation and
thrombophilia in liver disease Journal of
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Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Figure 1 Mean Plasma vWF (umoll) in patients Figure 2 Mean Plasma ADAMTS 13 activity in patients
Child Score
141210864
vW
F
600
500
400
300
200
100
0
Figure 3 Correlation between vWF and Child Pugh score in patients
Figure 5 correlation between ADAMTS 13 and Child Pugh Score in patients
Adamts 13
706050403020100
Ch
ild S
co
re
14
12
10
8
6
4
Figure 4 Correlation between ADAMTS 13 and Child Pugh score in patients
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
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42
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M Matsuyama T et al Localization of
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18 Suzuki M Murata M Matsubara Y Von
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313(1) 212-16
19 Turner N Nolasco L Tao Z Dong JF Moak J
Human endothelial cells synthesize and release
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Haemostasis 2006 4(6) 1396-404
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21 Martin P Friedman S Dienstag L Diagnostic
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Thomas C Viral hepatitis Scientific basis and
clinical management UK Longman Group 1993
393- 409
22 Sadler JE A new name in thrombosis
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552-4
23 Deng L Bremme K Hansson LO Blomback M
Plasma levels of von Willebrand factor and
fibronectin as markers of persisting endothelial
damage in preeclampsia Obstet Gynecol 1994
84(6) 941-5
24 Violi F Ferro D Basili S Quintarelli C Musca A
et al Hyperfibrinolysis resulting from clotting
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25 Adams DH Burra P Hubscher SG Elias E
Newman W Endothelial activation and circulating
vascular adhesion molecules in alcoholic liver
disease Hepatology 1994 19 588-94
26 Northup PG Sundaram V Fallon MB reddy KR
Balogun RA et al Hypercoagulation and
thrombophilia in liver disease Journal of
Thrombosis and Haemostasis 2008 6 2-9
27 Ferro D Quintarelli C Lattuada A Leo R
Alessandroni M et al High plasma levels of von
Willebrand factor as a marker of endothelial
perturbation in cirrhosis relationship to
endotoxaemia Hepatology 1996 23(6) 1377-83
28 Kulwas A Szaflarska SA Kotschy M czerwionka
SM et al Von Willebrand factor and
thrombpmodulin as markers of endothelial cell
functions in children with chronic viral hepatitis
Med Wieku Rozwoj 2004 8(1) 107-14 (abstract)
29 La Mura V reverter JC Arroyo AF Raffa S
Reverter E et al Von Wiilebrand levels predict
clinical outcome in patients with cirrhosis and
portal hypertension Gut 2011 60 1133-8
30 Albornoz L Alvarez D Otaso JC Gadano A
Salviu J et al Von Willebrand factor could be an
index of endothelial dysfunction in patients with
cirrhosis relationship to degree of liver failure and
nitric oxide levels J Hepatol 1999 38 451-5
31 Beer J Clerici N Baillod P Von Felten A
Schlappritzi E et al Quantitative and qualitative
analysis of platelet GpIb and von Willebrand
factor in liver cirrhosis Thromb Haemost 1995
73 601-9
32 Moake J Turner N Stathopouolos N Nolasco L
Hellums D et al Involvement of large von
Willebrand factor (vWF) multimers and unusually
larger vWF forms derived from endothelial cells in
shear stress induced platelet aggregation J Clin
Invest 1986 78 1456-61
33 Park YD Yoshioka A Kawa K Ishizashi H Yagi
H et al Impaired activity of plasma von
Willebrand factorcleaving protease may predict
the occurrence of hepatic veno-occlusive disease
after stem cell transplantation Bone Marrow
Transplantation 2002 29(9) 789-94
34 Uemura M Fujimura Y Matsuyama T Potential
role of ADAMTS13 in the progression of
alcoholic hepatitis Current Drug Abuse Reviews
2008 1(2) 188-96
35 Feys HB Canciani MT Peyvandi F Deckmyn H
Vanhoorellbeke K et al ADAMTS13 activity to
antigen ratio in physiological and pathological
conditions associated with an increased risk of
thrombosis British Journal of Haematology 2007
138(4) 534-40
36 Yagita M Uemura M Nakamura T Kunitomi A
Matsumoto M et al Development of ADAMTS13
inhibitor in a patient with hepatitis C virus-related
liver cirrhosis causes thrombotic
thrombocytopenic purpura Journal of Hepatology
2005 42(3) 420-1
37 Mannucci PM Canciani MT Forza I Changes in
health and disease of the metalloprotease that
cleaves vonWillebrand factor Blood 2001 98(9)
2730-5
38 Uemura M Fujimura Y Matsumoto M Ishizashi
H Kato S et al Comprehensive analysis of
ADAMTS13 in patients with liver cirrhosis
Thrombosis and Haemostasis 2008 99(6) 1019-
29
39 Matsumoto M Chisuwa H Nakazawa Y Living
related liver transplantation rescues reduced vWF-
cleaving protease activity in patients with cirrhotic
biliary atresia Blood 2000 96 636-40
40 Lisman T Bongers TN Adelmeijer J Janssen HL
Elevated levels of von Willebrand factor in
cirrhosis support platelet adhesion despite reduced
functional capacity Hepatology 2006 44 53-61
41 Umeura M Fujimura Y Ko S Matsumoto M
Nakajima Y et al Pivotal role of ADAMTS 13
function in liver diseases Int J Hematol 2010
91(1) 20-9
42 Bernardo A Ball C Nolasco L Moak JF Dong
JF Effects of inflammatory cytokines on the
release and cleavage of the endothelial cell-derived
ultralarge von Willebrand factor multimers under
flow Blood 2004 104(1) 100-6
43 Cao WJ Niiya M Zheng XW Shang DZ Zheng
XL Inflammatory cytokines inhibit ADAMTS13
synthesis in hepatic stellate cells and endothelial
cells Journal of Thrombosis and Haemostasis
2008 6(7) 1233-5
44 Furlan M Robles R Galbusera M Remuzzi G
Kyrle PA et al Von Willebrand factor-cleaving
protease in thrombotic thrombocytopenic purpura
and the hemolytic-uremic syndrome New Engl J
Med 1998 339(22) 1578-84
45 Tsai HM Lian EC Antibodies to von Willebrand
factor-cleaving protease in acute thrombotic
thrombocytopenic purpura New Engl J Med 1998
339(22) 1585-94
46 Kume Y Ikeda H Inoue M Tejima K Tomiya T
et al Hepatic stellate cell damage may lead to
decreased plasma ADAMTS13 activity in rats
FEBS Letters 2007 581(8) 1631-4
47 Niiya M Uemura M Zheng XW Pollak ES
Dockal M et al Increased ADAMTS-13
proteolytic activity in rat hepatic stellate cells upon
activation in vitro and in vivo Journal of
Thrombosis and Haemostasis 2006 4(5) 1063-70
48 Claus RA Bockmeyer CL Sossdorf M Losche
W The balance between von-Willebrand factor
and its cleaving protease ADAMTS13 biomarker
in systemic inflammation and development of
organ failure Current Molecular Medicine 2010
10(2) 236-48
49 Reiter RA Varadi K Turecek PL Jilma B Knobl
P Changes in ADAMTS13 (von-Willebrand-
factorcleaving protease) activity after induced
release of von Willebrand factor during acute
systemic inflammation Thrombosis and
Haemostasis 2005 93(3) 554-8
50 Ishikawa M Uemura M Matsuyama T
Matsumoto M Ishizashi H et al Potential role of
enhanced cytokinemia and plasma inhibitor on the
decreased activity of plasma ADAMTS13 in
patients with alcoholic hepatitis relationship to
endotoxemia Alcoholism Clinical and
Experimental Research 2010 34(1) 25-33
51 Amitrano L Guardascione MA Brancaccio V
Margaglione M Manguso F et al Risk factors and
clinical presentation of portal vein thrombosis in
patients with liver cirrhosis Journal of Hepatology
2004 40(5) 736-41
52 Wanless IR Wong F Blendis LM Greig P
Heathcote EJ et al Hepatic and portal vein
thrombosis in cirrhosis possible role in
development of parenchymal extinction and portal
hypertension Hepatology 1995 21(5) 1238-47
53 Pluta A Gutkowski K Hartleb M Coagulopathy
in liver diseases Advanced Medical Science 2010
55 16-21
54 Banno F Kokame K Okuda T Honda S Miyata S
et al Complete deficiency in ADAMTS13 is
prothrombotic but alone is not sufficient to cause
thrombotic thrombocytopenic purpura Blood
2006 107 3161-6
Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Discussion
Liver cirrhosis is frequently accompanied by
complex alterations in the hemostatic system
resulting in bleeding tendency Although
many hemostatic changes in liver disease
promote bleeding compensatory mechanisms
are found (24) There is indirect evidence that
there may be an endothelial perturbation in
liver cirrhosis which might explain some of
the clinical and biochemical changes as well
as the hyperdynamic circulation and
coagulation changes seen in cirrhosis (25)
Considering that ADAMTS 13 is synthesized
in HSCs and ULvWFMs is produced in
transformed sinusoidal endothelial cells
(SEC) during liver injury decreased plasma
ADAMTS13 may involve not only sinusoidal
microcirculatory disturbances but also
subsequent progression of liver disease
finally leading to multiorgan failure (26) In the
present work mean value of vWF was
significantly higher in liver cirrhotic patients
than in controls Similar findings were
reported by ferro et al (27) Kulwas et al (28)
and La Mura et al (29) Our findings support
the hypothesis of endothelial activation in
liver cirrhosis In the present study
significant positive correlation was found
between plasma vWF and Child Pugh
scoreSimilar findings were reported by La
Mura et al ( 29) Albornoz et al(30)demonstrated
that the increased plasma levels of vWF
paralleled the degree of liver failure (30) High
circulating levels of vWF in cirrhosis might
be a consequence of endothelial perturbation
induced by endotoxin Ferro et al reported a
strong correlation between endotoxemia and
high circulating levels of vWF(27) Increased
serum levels of vWF could be due to
endothelial activation by cytokines and or
endothelial damage (27) Also it has been
suggested that high vWF levels can be
probably explained by increased synthesis
release of this protein since it is an acute
phase reactant to tissue injury Furthermore
high vWF levels could be associated with a
compensatory regulation mechanism for the
hemostatic disorders of chronic liver disease (31) Another hypothesis of high vWF is that
local damage to endothelial cells could result
in the disruption of Weibel- Palade bodies and
endothelial membrane leading to vWF
multimers release (32) Studies have shown
that ADAMTS13 is significantly decreased in
patients with hepatic veno-occlusive disease
(VOD) (33) alcoholic hepatitis(34) liver
cirrhosis (35) Furthermore HCV related liver
cirrhosis patients with ADAMTS13 inhibitors
typically developed thrombotic thrombo-
cytopenic purpura (TTP) (36) In the present
work plasma ADAMTS13 activity was
significantly lowered in patients with liver
cirrhosis than in controls Furthermore in
patients with liver cirrhosis plasma
ADAMTS 13 was significantly lower in Child
C patients than in Child B and A patients and
also in Child B patients than Child A patients
In our work ADAMTS13 decreased signify-
cantly with advanced cirrhotic process by the
observed negative correlation between
ADAMTS13 and Child Pugh Score Our
results were in agreement with that of
Mannucci et al (37) who reported a reduction
in ADAMTS13 activity in advanced liver
cirrhosis and the decrease of plasma
ADAMTS 13 activity in patients with chronic
liver disease has been associated with disease
progression In addition Uemeura et al (38)
reported that both ADAMTS13 and its
antigen decreased in cirrhotic patients and
such decrease was positively correlated with
increasing severity of cirrhosis assessed by
Child Pugh criteria Moreover Matsumoto et
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
References
1 Houghton M The long and winding road leading
to the identification of the hepatitis C virus
Journal of Hepatology 2009 5 939-48
2 El Zanaty F Way A Egypt demographic and
health survey 2008 Cairo Egypt Ministry of
Health EL- Zanaty and associates and Macro
International 2009
3 Wilkins T Malcolm JK Raina D Hepatitis C
diagnosis and treatment American family
physician 2010 81(11) 1351-7
4 Ragni MV Belle SH Impact of human
immunodeficiency virus infection on progression
to end-stage liver disease in individuals with
hemophilia and hepatitis C virus infection J Infect
Dis 2001 183(7) 1112-5
5 Kumar V Abbas AK Faousto N Robbins and
Cotran Pathologic basis of disease 2005 (7)
Philadelphia Elsevier
6 Lisman T Leebeek FW de Groot PG
Haemostatic abnormalities in patients with liver
disease J Hepatol 2002 37 280-7
7 Furie B Furie BC Thrombus formation in vivo J
Clin Invest 2005 115(12) 3355-62
8 Pallister CJ Watson MS Haematology Scion
Publishing 2010 2 334-6
9 Schaffner F Popper H Capillarization of hepatic
sinusoids in man Gastroenterology 1963 44 239-
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Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
al (39) demonstrated that decreased plasma
ADAMTS13 in patients with cirrhotic biliary
atresia can be fully restored after liver
transplantation indicating that the liver is the
main organ producing ADAMTS13 On the
other hand Lisman et al (40) showed that
plasma ADAMTS13 and its antigen were not
significantly different between various stages
of liver cirrhosis The discrepancy between
our results and that of Lismanrsquos is attributed
to the fact that all of our cirrhotic patients are
secondary to chronic HCV infection whereas
in Lismanrsquos study half of the cirrhotic
patients were alcoholic The mechanism
responsible for the decrease in ADAMTS13
in advanced liver cirrhosis may include
enhanced consumption due to the degradation
of large quantities of vWF (41) inflammatory
cytokines(4243) andor ADAMTS13 plasma
inhibitor(4445) It is controversial whether
ADAMTS 13 deficiency is caused by
decreased production in the liver Some
studies have shown that HSC apoptosis plays
an essential role in decreased ADAMTS13
(46) Other studies demonstrated the presence
of the inhibitor to ADAMTS 13 in 83 of
patients with moderate to severe ADAMTS
13 deficiency (47)Alternatively cytokinemia (48) and endotoxemia (49) are additional
potential candidates for decreasing plasma
ADAMTS13 Recent investigations demon-
strated that IL-6 inhibited the action of
ADAMTS13 and both IL-8 and TNF-α
stimulated the release of ULvWFMs in
human umbilical vein endothelial cells in
vitro (42) IFN-γ IL-4 and TNF- α also inhibit
ADAMTS13 synthesis and activity in rat
primary HSC (43) To the best of our
knowledge no available studies in literature
demonstrated the relationship between
ADAMTS 13 and vWF in HCV induced liver
cirrhosis in Egyptian patients so we have
tried to explore this point in the current study
Our results revealed significant ndashve
correlation between ADAMTs 13 and vWF in
the studied patients group this was supported
by the study that IV infusion of endotoxin to
healthy volunteers caused a decrease in
plasma ADAMTS 13 together with the
appearance of ULvWFMs (49) Additionally
in patients with alcoholic hepatitis especially
in severe cases complicated with liver
cirrhosis ADAMTS 13 is concomitantly
decreased and vWF antigen is progressively
increased with increasing concentration of IL-
6 and IL-8 above 100pgml (50) The
relationship between ADAMTS 13 and vWF
in the above mentioned studies supports our
finding of the presence of significant negative
correlation between vWF and ADAMTS 13
Patients with advanced liver cirrhosis may
frequently develop hepatic and portal vein
thrombosis (5152) Such a hyper-coagulable
state in liver diseases may be involved in
hepatic parenchymal destruction acceleration
of liver fibrosis and disease progression
leading to hepatorenal syndrome porto-
pulmonary hypertension and spontaneous
bacterial peritonitis (SBP) (53) In our work
plasma ADAMTS 13 was significantly
lowered in those with portal vein thrombosis
(10 patients) than in those without it (30
patients) On the other hand high vWF levels
were noticed in those with portal vein
thrombosis This observation was in agree-
ment with that of Uemura et al (38) who
propose that ADAMTS13 deficiency might
contribute to the development of portal vein
thrombosis in patients with advanced
cirrhosis Although ADAMTS13 deficiency
creates a prethrombotic state it may not
appear because of the presence of
thrombocytopenia and coagulation factor
deficiencies in liver cirrhosis (54)
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
References
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to the identification of the hepatitis C virus
Journal of Hepatology 2009 5 939-48
2 El Zanaty F Way A Egypt demographic and
health survey 2008 Cairo Egypt Ministry of
Health EL- Zanaty and associates and Macro
International 2009
3 Wilkins T Malcolm JK Raina D Hepatitis C
diagnosis and treatment American family
physician 2010 81(11) 1351-7
4 Ragni MV Belle SH Impact of human
immunodeficiency virus infection on progression
to end-stage liver disease in individuals with
hemophilia and hepatitis C virus infection J Infect
Dis 2001 183(7) 1112-5
5 Kumar V Abbas AK Faousto N Robbins and
Cotran Pathologic basis of disease 2005 (7)
Philadelphia Elsevier
6 Lisman T Leebeek FW de Groot PG
Haemostatic abnormalities in patients with liver
disease J Hepatol 2002 37 280-7
7 Furie B Furie BC Thrombus formation in vivo J
Clin Invest 2005 115(12) 3355-62
8 Pallister CJ Watson MS Haematology Scion
Publishing 2010 2 334-6
9 Schaffner F Popper H Capillarization of hepatic
sinusoids in man Gastroenterology 1963 44 239-
42
10 Martell M Coll M Raurell I Ezkurdia N Raurell
I et al Pathophysiology of splanchnic
vasodilatation in portal hypertension World J
Hepatol 2010 2(6) 208-20
11 Albers I Hartman H Bircher J Superiority of the
Child Pugh classification to quantitative liver
function tests for assessing prognosis of liver
cirrhosis Scand J Gastroenterol 1989 24 269-76
12 Ruggeri ZM Ware J The structure and function of
von Willebrand factor Thromb Haemost 1992 67
594-8
13 Moake JL Thrombotic microangiopathies New
Engl J M 2002 347(8) 589-600
14 Fujimura Y Matsumoto M Yagi H Yoshioka A
Matsui T et al Von Willebrand factor-cleaving
protease and Upshaw-Schulman syndrome Int J of
Hematol 2002 75(1) 25-34
15 Padilla A Moake JL Bernardo A Ball C Wang Y
et al P-selectin anchors newly released ultralarge
von Willebrand factor multimers to the endothelial
cell surface Blood 2004 103(6) 2150-6
16 Emsley J Cruz M Handin R Liddington R
Crystal structure of the von Willebrand factor A1
domain and implication for the binding of platelet
glycoprotein 1b J Biol chem 1998 273 361-401
17 Uemura M Tatsumi K Matsumoto M Fujimoto
M Matsuyama T et al Localization of
ADAMTS13 to the stellate cells of human liver
Blood 2005 106(3) 922-24
18 Suzuki M Murata M Matsubara Y Von
Willebrand factor-cleaving protease (ADAMTS-
13) in human platelets Biochemical and
Biophysical Research Communications 2004
313(1) 212-16
19 Turner N Nolasco L Tao Z Dong JF Moak J
Human endothelial cells synthesize and release
ADAMTS- 13 Journal of Thrombosis and
Haemostasis 2006 4(6) 1396-404
20 Burits H Ashwood R Tietz fundamentals of
clinical chemistry 4th ed Philadelphia Saunders
WB 1996 82-5
21 Martin P Friedman S Dienstag L Diagnostic
approach to viral hepatitis In Zuckerman J
Thomas C Viral hepatitis Scientific basis and
clinical management UK Longman Group 1993
393- 409
22 Sadler JE A new name in thrombosis
ADAMTS13 Proc Natl Acad Sci U S A 2002 99
552-4
23 Deng L Bremme K Hansson LO Blomback M
Plasma levels of von Willebrand factor and
fibronectin as markers of persisting endothelial
damage in preeclampsia Obstet Gynecol 1994
84(6) 941-5
24 Violi F Ferro D Basili S Quintarelli C Musca A
et al Hyperfibrinolysis resulting from clotting
activation in patients with different stages of liver
cirrhosis Hepatology 1993 17 78-83
25 Adams DH Burra P Hubscher SG Elias E
Newman W Endothelial activation and circulating
vascular adhesion molecules in alcoholic liver
disease Hepatology 1994 19 588-94
26 Northup PG Sundaram V Fallon MB reddy KR
Balogun RA et al Hypercoagulation and
thrombophilia in liver disease Journal of
Thrombosis and Haemostasis 2008 6 2-9
27 Ferro D Quintarelli C Lattuada A Leo R
Alessandroni M et al High plasma levels of von
Willebrand factor as a marker of endothelial
perturbation in cirrhosis relationship to
endotoxaemia Hepatology 1996 23(6) 1377-83
28 Kulwas A Szaflarska SA Kotschy M czerwionka
SM et al Von Willebrand factor and
thrombpmodulin as markers of endothelial cell
functions in children with chronic viral hepatitis
Med Wieku Rozwoj 2004 8(1) 107-14 (abstract)
29 La Mura V reverter JC Arroyo AF Raffa S
Reverter E et al Von Wiilebrand levels predict
clinical outcome in patients with cirrhosis and
portal hypertension Gut 2011 60 1133-8
30 Albornoz L Alvarez D Otaso JC Gadano A
Salviu J et al Von Willebrand factor could be an
index of endothelial dysfunction in patients with
cirrhosis relationship to degree of liver failure and
nitric oxide levels J Hepatol 1999 38 451-5
31 Beer J Clerici N Baillod P Von Felten A
Schlappritzi E et al Quantitative and qualitative
analysis of platelet GpIb and von Willebrand
factor in liver cirrhosis Thromb Haemost 1995
73 601-9
32 Moake J Turner N Stathopouolos N Nolasco L
Hellums D et al Involvement of large von
Willebrand factor (vWF) multimers and unusually
larger vWF forms derived from endothelial cells in
shear stress induced platelet aggregation J Clin
Invest 1986 78 1456-61
33 Park YD Yoshioka A Kawa K Ishizashi H Yagi
H et al Impaired activity of plasma von
Willebrand factorcleaving protease may predict
the occurrence of hepatic veno-occlusive disease
after stem cell transplantation Bone Marrow
Transplantation 2002 29(9) 789-94
34 Uemura M Fujimura Y Matsuyama T Potential
role of ADAMTS13 in the progression of
alcoholic hepatitis Current Drug Abuse Reviews
2008 1(2) 188-96
35 Feys HB Canciani MT Peyvandi F Deckmyn H
Vanhoorellbeke K et al ADAMTS13 activity to
antigen ratio in physiological and pathological
conditions associated with an increased risk of
thrombosis British Journal of Haematology 2007
138(4) 534-40
36 Yagita M Uemura M Nakamura T Kunitomi A
Matsumoto M et al Development of ADAMTS13
inhibitor in a patient with hepatitis C virus-related
liver cirrhosis causes thrombotic
thrombocytopenic purpura Journal of Hepatology
2005 42(3) 420-1
37 Mannucci PM Canciani MT Forza I Changes in
health and disease of the metalloprotease that
cleaves vonWillebrand factor Blood 2001 98(9)
2730-5
38 Uemura M Fujimura Y Matsumoto M Ishizashi
H Kato S et al Comprehensive analysis of
ADAMTS13 in patients with liver cirrhosis
Thrombosis and Haemostasis 2008 99(6) 1019-
29
39 Matsumoto M Chisuwa H Nakazawa Y Living
related liver transplantation rescues reduced vWF-
cleaving protease activity in patients with cirrhotic
biliary atresia Blood 2000 96 636-40
40 Lisman T Bongers TN Adelmeijer J Janssen HL
Elevated levels of von Willebrand factor in
cirrhosis support platelet adhesion despite reduced
functional capacity Hepatology 2006 44 53-61
41 Umeura M Fujimura Y Ko S Matsumoto M
Nakajima Y et al Pivotal role of ADAMTS 13
function in liver diseases Int J Hematol 2010
91(1) 20-9
42 Bernardo A Ball C Nolasco L Moak JF Dong
JF Effects of inflammatory cytokines on the
release and cleavage of the endothelial cell-derived
ultralarge von Willebrand factor multimers under
flow Blood 2004 104(1) 100-6
43 Cao WJ Niiya M Zheng XW Shang DZ Zheng
XL Inflammatory cytokines inhibit ADAMTS13
synthesis in hepatic stellate cells and endothelial
cells Journal of Thrombosis and Haemostasis
2008 6(7) 1233-5
44 Furlan M Robles R Galbusera M Remuzzi G
Kyrle PA et al Von Willebrand factor-cleaving
protease in thrombotic thrombocytopenic purpura
and the hemolytic-uremic syndrome New Engl J
Med 1998 339(22) 1578-84
45 Tsai HM Lian EC Antibodies to von Willebrand
factor-cleaving protease in acute thrombotic
thrombocytopenic purpura New Engl J Med 1998
339(22) 1585-94
46 Kume Y Ikeda H Inoue M Tejima K Tomiya T
et al Hepatic stellate cell damage may lead to
decreased plasma ADAMTS13 activity in rats
FEBS Letters 2007 581(8) 1631-4
47 Niiya M Uemura M Zheng XW Pollak ES
Dockal M et al Increased ADAMTS-13
proteolytic activity in rat hepatic stellate cells upon
activation in vitro and in vivo Journal of
Thrombosis and Haemostasis 2006 4(5) 1063-70
48 Claus RA Bockmeyer CL Sossdorf M Losche
W The balance between von-Willebrand factor
and its cleaving protease ADAMTS13 biomarker
in systemic inflammation and development of
organ failure Current Molecular Medicine 2010
10(2) 236-48
49 Reiter RA Varadi K Turecek PL Jilma B Knobl
P Changes in ADAMTS13 (von-Willebrand-
factorcleaving protease) activity after induced
release of von Willebrand factor during acute
systemic inflammation Thrombosis and
Haemostasis 2005 93(3) 554-8
50 Ishikawa M Uemura M Matsuyama T
Matsumoto M Ishizashi H et al Potential role of
enhanced cytokinemia and plasma inhibitor on the
decreased activity of plasma ADAMTS13 in
patients with alcoholic hepatitis relationship to
endotoxemia Alcoholism Clinical and
Experimental Research 2010 34(1) 25-33
51 Amitrano L Guardascione MA Brancaccio V
Margaglione M Manguso F et al Risk factors and
clinical presentation of portal vein thrombosis in
patients with liver cirrhosis Journal of Hepatology
2004 40(5) 736-41
52 Wanless IR Wong F Blendis LM Greig P
Heathcote EJ et al Hepatic and portal vein
thrombosis in cirrhosis possible role in
development of parenchymal extinction and portal
hypertension Hepatology 1995 21(5) 1238-47
53 Pluta A Gutkowski K Hartleb M Coagulopathy
in liver diseases Advanced Medical Science 2010
55 16-21
54 Banno F Kokame K Okuda T Honda S Miyata S
et al Complete deficiency in ADAMTS13 is
prothrombotic but alone is not sufficient to cause
thrombotic thrombocytopenic purpura Blood
2006 107 3161-6
Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Conclusion
Egyptian cirrhotic chronic HCV patients may
experience a hemostatic disorders which are
evidenced by high levels of vWF and
concomitant low levels of ADAMTS 13
suggesting the possible role of both
endothelial and liver dysfunction in those
patients Moreover further studies are needed
to explore different therapeutic areas aiming
at elevating levels of ADAMTS 13
particularly in those patients with portal vein
thrombosis
References
1 Houghton M The long and winding road leading
to the identification of the hepatitis C virus
Journal of Hepatology 2009 5 939-48
2 El Zanaty F Way A Egypt demographic and
health survey 2008 Cairo Egypt Ministry of
Health EL- Zanaty and associates and Macro
International 2009
3 Wilkins T Malcolm JK Raina D Hepatitis C
diagnosis and treatment American family
physician 2010 81(11) 1351-7
4 Ragni MV Belle SH Impact of human
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to end-stage liver disease in individuals with
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Dis 2001 183(7) 1112-5
5 Kumar V Abbas AK Faousto N Robbins and
Cotran Pathologic basis of disease 2005 (7)
Philadelphia Elsevier
6 Lisman T Leebeek FW de Groot PG
Haemostatic abnormalities in patients with liver
disease J Hepatol 2002 37 280-7
7 Furie B Furie BC Thrombus formation in vivo J
Clin Invest 2005 115(12) 3355-62
8 Pallister CJ Watson MS Haematology Scion
Publishing 2010 2 334-6
9 Schaffner F Popper H Capillarization of hepatic
sinusoids in man Gastroenterology 1963 44 239-
42
10 Martell M Coll M Raurell I Ezkurdia N Raurell
I et al Pathophysiology of splanchnic
vasodilatation in portal hypertension World J
Hepatol 2010 2(6) 208-20
11 Albers I Hartman H Bircher J Superiority of the
Child Pugh classification to quantitative liver
function tests for assessing prognosis of liver
cirrhosis Scand J Gastroenterol 1989 24 269-76
12 Ruggeri ZM Ware J The structure and function of
von Willebrand factor Thromb Haemost 1992 67
594-8
13 Moake JL Thrombotic microangiopathies New
Engl J M 2002 347(8) 589-600
14 Fujimura Y Matsumoto M Yagi H Yoshioka A
Matsui T et al Von Willebrand factor-cleaving
protease and Upshaw-Schulman syndrome Int J of
Hematol 2002 75(1) 25-34
15 Padilla A Moake JL Bernardo A Ball C Wang Y
et al P-selectin anchors newly released ultralarge
von Willebrand factor multimers to the endothelial
cell surface Blood 2004 103(6) 2150-6
16 Emsley J Cruz M Handin R Liddington R
Crystal structure of the von Willebrand factor A1
domain and implication for the binding of platelet
glycoprotein 1b J Biol chem 1998 273 361-401
17 Uemura M Tatsumi K Matsumoto M Fujimoto
M Matsuyama T et al Localization of
ADAMTS13 to the stellate cells of human liver
Blood 2005 106(3) 922-24
18 Suzuki M Murata M Matsubara Y Von
Willebrand factor-cleaving protease (ADAMTS-
13) in human platelets Biochemical and
Biophysical Research Communications 2004
313(1) 212-16
19 Turner N Nolasco L Tao Z Dong JF Moak J
Human endothelial cells synthesize and release
ADAMTS- 13 Journal of Thrombosis and
Haemostasis 2006 4(6) 1396-404
20 Burits H Ashwood R Tietz fundamentals of
clinical chemistry 4th ed Philadelphia Saunders
WB 1996 82-5
21 Martin P Friedman S Dienstag L Diagnostic
approach to viral hepatitis In Zuckerman J
Thomas C Viral hepatitis Scientific basis and
clinical management UK Longman Group 1993
393- 409
22 Sadler JE A new name in thrombosis
ADAMTS13 Proc Natl Acad Sci U S A 2002 99
552-4
23 Deng L Bremme K Hansson LO Blomback M
Plasma levels of von Willebrand factor and
fibronectin as markers of persisting endothelial
damage in preeclampsia Obstet Gynecol 1994
84(6) 941-5
24 Violi F Ferro D Basili S Quintarelli C Musca A
et al Hyperfibrinolysis resulting from clotting
activation in patients with different stages of liver
cirrhosis Hepatology 1993 17 78-83
25 Adams DH Burra P Hubscher SG Elias E
Newman W Endothelial activation and circulating
vascular adhesion molecules in alcoholic liver
disease Hepatology 1994 19 588-94
26 Northup PG Sundaram V Fallon MB reddy KR
Balogun RA et al Hypercoagulation and
thrombophilia in liver disease Journal of
Thrombosis and Haemostasis 2008 6 2-9
27 Ferro D Quintarelli C Lattuada A Leo R
Alessandroni M et al High plasma levels of von
Willebrand factor as a marker of endothelial
perturbation in cirrhosis relationship to
endotoxaemia Hepatology 1996 23(6) 1377-83
28 Kulwas A Szaflarska SA Kotschy M czerwionka
SM et al Von Willebrand factor and
thrombpmodulin as markers of endothelial cell
functions in children with chronic viral hepatitis
Med Wieku Rozwoj 2004 8(1) 107-14 (abstract)
29 La Mura V reverter JC Arroyo AF Raffa S
Reverter E et al Von Wiilebrand levels predict
clinical outcome in patients with cirrhosis and
portal hypertension Gut 2011 60 1133-8
30 Albornoz L Alvarez D Otaso JC Gadano A
Salviu J et al Von Willebrand factor could be an
index of endothelial dysfunction in patients with
cirrhosis relationship to degree of liver failure and
nitric oxide levels J Hepatol 1999 38 451-5
31 Beer J Clerici N Baillod P Von Felten A
Schlappritzi E et al Quantitative and qualitative
analysis of platelet GpIb and von Willebrand
factor in liver cirrhosis Thromb Haemost 1995
73 601-9
32 Moake J Turner N Stathopouolos N Nolasco L
Hellums D et al Involvement of large von
Willebrand factor (vWF) multimers and unusually
larger vWF forms derived from endothelial cells in
shear stress induced platelet aggregation J Clin
Invest 1986 78 1456-61
33 Park YD Yoshioka A Kawa K Ishizashi H Yagi
H et al Impaired activity of plasma von
Willebrand factorcleaving protease may predict
the occurrence of hepatic veno-occlusive disease
after stem cell transplantation Bone Marrow
Transplantation 2002 29(9) 789-94
34 Uemura M Fujimura Y Matsuyama T Potential
role of ADAMTS13 in the progression of
alcoholic hepatitis Current Drug Abuse Reviews
2008 1(2) 188-96
35 Feys HB Canciani MT Peyvandi F Deckmyn H
Vanhoorellbeke K et al ADAMTS13 activity to
antigen ratio in physiological and pathological
conditions associated with an increased risk of
thrombosis British Journal of Haematology 2007
138(4) 534-40
36 Yagita M Uemura M Nakamura T Kunitomi A
Matsumoto M et al Development of ADAMTS13
inhibitor in a patient with hepatitis C virus-related
liver cirrhosis causes thrombotic
thrombocytopenic purpura Journal of Hepatology
2005 42(3) 420-1
37 Mannucci PM Canciani MT Forza I Changes in
health and disease of the metalloprotease that
cleaves vonWillebrand factor Blood 2001 98(9)
2730-5
38 Uemura M Fujimura Y Matsumoto M Ishizashi
H Kato S et al Comprehensive analysis of
ADAMTS13 in patients with liver cirrhosis
Thrombosis and Haemostasis 2008 99(6) 1019-
29
39 Matsumoto M Chisuwa H Nakazawa Y Living
related liver transplantation rescues reduced vWF-
cleaving protease activity in patients with cirrhotic
biliary atresia Blood 2000 96 636-40
40 Lisman T Bongers TN Adelmeijer J Janssen HL
Elevated levels of von Willebrand factor in
cirrhosis support platelet adhesion despite reduced
functional capacity Hepatology 2006 44 53-61
41 Umeura M Fujimura Y Ko S Matsumoto M
Nakajima Y et al Pivotal role of ADAMTS 13
function in liver diseases Int J Hematol 2010
91(1) 20-9
42 Bernardo A Ball C Nolasco L Moak JF Dong
JF Effects of inflammatory cytokines on the
release and cleavage of the endothelial cell-derived
ultralarge von Willebrand factor multimers under
flow Blood 2004 104(1) 100-6
43 Cao WJ Niiya M Zheng XW Shang DZ Zheng
XL Inflammatory cytokines inhibit ADAMTS13
synthesis in hepatic stellate cells and endothelial
cells Journal of Thrombosis and Haemostasis
2008 6(7) 1233-5
44 Furlan M Robles R Galbusera M Remuzzi G
Kyrle PA et al Von Willebrand factor-cleaving
protease in thrombotic thrombocytopenic purpura
and the hemolytic-uremic syndrome New Engl J
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339(22) 1585-94
46 Kume Y Ikeda H Inoue M Tejima K Tomiya T
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48 Claus RA Bockmeyer CL Sossdorf M Losche
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50 Ishikawa M Uemura M Matsuyama T
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52 Wanless IR Wong F Blendis LM Greig P
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53 Pluta A Gutkowski K Hartleb M Coagulopathy
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54 Banno F Kokame K Okuda T Honda S Miyata S
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Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
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Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
38 Uemura M Fujimura Y Matsumoto M Ishizashi
H Kato S et al Comprehensive analysis of
ADAMTS13 in patients with liver cirrhosis
Thrombosis and Haemostasis 2008 99(6) 1019-
29
39 Matsumoto M Chisuwa H Nakazawa Y Living
related liver transplantation rescues reduced vWF-
cleaving protease activity in patients with cirrhotic
biliary atresia Blood 2000 96 636-40
40 Lisman T Bongers TN Adelmeijer J Janssen HL
Elevated levels of von Willebrand factor in
cirrhosis support platelet adhesion despite reduced
functional capacity Hepatology 2006 44 53-61
41 Umeura M Fujimura Y Ko S Matsumoto M
Nakajima Y et al Pivotal role of ADAMTS 13
function in liver diseases Int J Hematol 2010
91(1) 20-9
42 Bernardo A Ball C Nolasco L Moak JF Dong
JF Effects of inflammatory cytokines on the
release and cleavage of the endothelial cell-derived
ultralarge von Willebrand factor multimers under
flow Blood 2004 104(1) 100-6
43 Cao WJ Niiya M Zheng XW Shang DZ Zheng
XL Inflammatory cytokines inhibit ADAMTS13
synthesis in hepatic stellate cells and endothelial
cells Journal of Thrombosis and Haemostasis
2008 6(7) 1233-5
44 Furlan M Robles R Galbusera M Remuzzi G
Kyrle PA et al Von Willebrand factor-cleaving
protease in thrombotic thrombocytopenic purpura
and the hemolytic-uremic syndrome New Engl J
Med 1998 339(22) 1578-84
45 Tsai HM Lian EC Antibodies to von Willebrand
factor-cleaving protease in acute thrombotic
thrombocytopenic purpura New Engl J Med 1998
339(22) 1585-94
46 Kume Y Ikeda H Inoue M Tejima K Tomiya T
et al Hepatic stellate cell damage may lead to
decreased plasma ADAMTS13 activity in rats
FEBS Letters 2007 581(8) 1631-4
47 Niiya M Uemura M Zheng XW Pollak ES
Dockal M et al Increased ADAMTS-13
proteolytic activity in rat hepatic stellate cells upon
activation in vitro and in vivo Journal of
Thrombosis and Haemostasis 2006 4(5) 1063-70
48 Claus RA Bockmeyer CL Sossdorf M Losche
W The balance between von-Willebrand factor
and its cleaving protease ADAMTS13 biomarker
in systemic inflammation and development of
organ failure Current Molecular Medicine 2010
10(2) 236-48
49 Reiter RA Varadi K Turecek PL Jilma B Knobl
P Changes in ADAMTS13 (von-Willebrand-
factorcleaving protease) activity after induced
release of von Willebrand factor during acute
systemic inflammation Thrombosis and
Haemostasis 2005 93(3) 554-8
50 Ishikawa M Uemura M Matsuyama T
Matsumoto M Ishizashi H et al Potential role of
enhanced cytokinemia and plasma inhibitor on the
decreased activity of plasma ADAMTS13 in
patients with alcoholic hepatitis relationship to
endotoxemia Alcoholism Clinical and
Experimental Research 2010 34(1) 25-33
51 Amitrano L Guardascione MA Brancaccio V
Margaglione M Manguso F et al Risk factors and
clinical presentation of portal vein thrombosis in
patients with liver cirrhosis Journal of Hepatology
2004 40(5) 736-41
52 Wanless IR Wong F Blendis LM Greig P
Heathcote EJ et al Hepatic and portal vein
thrombosis in cirrhosis possible role in
development of parenchymal extinction and portal
hypertension Hepatology 1995 21(5) 1238-47
53 Pluta A Gutkowski K Hartleb M Coagulopathy
in liver diseases Advanced Medical Science 2010
55 16-21
54 Banno F Kokame K Okuda T Honda S Miyata S
et al Complete deficiency in ADAMTS13 is
prothrombotic but alone is not sufficient to cause
thrombotic thrombocytopenic purpura Blood
2006 107 3161-6
Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Original Article
Last Minute Donor Exclusion in Living Donor Liver Transplantation Impact
on Evaluation Program
Hany Shoreem1 Hossam Eldeen Soliman1 Osama Hegazy1 Sherief Saleh1 Wael Abdelrazek2 Nirmeen Fayed3 Taha
Yassen1 Kalied Abuelella1 Tarek Ibrahim1 Ibrahim Marwan1
1Department of HBP Surgery National Liver Istitute Egypt 2Department of Hepatology National Liver Institute
Egypt 3Department of Anaesthesia National Liver Institute Egypt
ABSTRACT
Minimizing the risk imposed to a healthy donor is one of the complex aspects of living donor liver transplantation
(LDLT) and implies a highly scrutinized evaluation process Nevertheless late events could lead to exclusion of some
of those evaluated donors Aim To investigate the late causes of exclusion of a potential LDLT donor and how far
these reasons contributed to changing our donor evaluation program Method Throughout 10 years more than 2500
potential living donors had been evaluated for liver donation for about 1500 potential recipients who presented for
LDLT at transplantation unit National Liver Institute (NLI) Menoufyia university Egypt from them only 194 were
transplanted The evaluation records of those donors were retrospectively analyzed for causes of late exclusion
Results Only 15 of the evaluated potential donors were accepted for donation and only 78 were donated
Exclusion reasons included late psychological instability in 4 donors and family pressure to withdraw consent in one
donor substance abuse in 2 donors Early pregnancy was discovered in one female donor week pre-transplantation The
pre-operatively revised blood group of another donor was discovered to be incompatible as the previously determined
blood group was wrong The presence of Factor V Leiden homozygous mutation was diagnosed in 2 donors In four
cases LDLT was aborted after donor laparotomy due to macroscopic diffuse hepatic changes considering the liver
unsuitable for donation One donor was convicted and imprisoned 2 weeks before LDLT Most of these exclusions
evoked discussions led to modifications in the evaluation protocol in addition to its impact on both donors and
recipients Conclusion Late pre-transplantation donor exclusion had its impact in donors recipients and resources
Reassessment of the donor evaluation protocol should be a dynamic process and it found to be greatly affected by these
late exclusions
Introduction
Donor safety is the most crucial aspect of
LDLT programs The aim of donor evaluation
protocols is to completely avoid donor
mortality and minimize both the incidence
and degree of donor morbidity Living liver
donation is associated with a small but real
possibility of mortality that may approach 05
(1 2) Due to the high costs most centers
have developed a stepwise evaluation
program to identify contraindications to
donation as early as possible (3) The
published donor evaluation protocols are all
very similar Most potential donors are
excluded based on the initial studies to rule
out underlying conditions that represent
increased surgical risk Immediate exclusion
criteria include donors who are currently
pregnant less than 18-year-old or older than
55-years old with co-morbidities Selection
criteria are rigid to ensure donor safety with
no exception to accommodate the needs of the
recipients (4) Acceptance rate for donors
undergoing the evaluation process is reported
to vary between 22 and 66 Taken into
consideration that not all recipients qualify for
LDLT and that not all potential recipients can
present a possibly suitable donor the chances
for a LDLT candidate to receive a living
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
donor graft are quite low and that only 13
of prospective recipients were able to find
suitable living donors(5 6) Other authors
reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of whom 15 were finally accepted(7)
Throughout the previous 10 years we faced
some problems and pitfalls during donorsrsquo
evaluation that led to their late exclusion from
donation soon before transplantation This
exclusion had effect on donor recipient
resources and the evaluation protocol
therefore some modifications had been raised
in the four steps of our protocol The aim of
this study is to investigate the late causes of
exclusion of a potential LDLT donor and how
far these reasons contributed to changing our
donor evaluation program
Patients and Methods
Throughout 10 years (from start of
transplantation program in April 2003 till
October 2012) more than 2500 potential
living donors had been evaluated for
suitability for liver donation for about 1500
potential recipients who presented for LDLT
at National Liver Institute Menoufyia
university from them only 194 finally
donated for LDLT after proceeding all of the
phases of donor selection in our protocol The
evaluation protocol that had been adopted at
the start of the transplantation program in our
center on April 2003 is illustrated in table 1
The donors were evaluated in four steps Step
one of this evaluation included a clinical and
social evaluation A liver profile hepatitis
marker assessment renal profile complete
blood count and abdominal ultrasound with
Doppler were performed in this step Step two
included an ultrasound-guided liver biopsy
Step two also included an imaging and
evaluation for cardiopulmonary status Step
three included an imaging evaluation of the
liver vascular anatomy using computed
tomography (CT) angiogram and imaging
evaluation of biliary anatomy and using
magnetic resonance cholangiopancreatogra-
phy A CT volumetric study was used to
calculate the volume of the liver parenchyma
Step three also included a screening of
procoagulation disorders Step four consisted
of final medical and anesthesia evaluation
blood preparation final psychological and
ethical evaluation scheduling of the surgical
date and consent of the donor
Table 1 Initial NLI stepwise donor evaluation protocol
Step 1
Clinical evaluation Medical history and physical examination relation to recipient age weight and size medical
history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and pancreas
profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV HSV
Abdominal ultrasound
Step 2
Liver biopsy
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography Pulmonary function
test Echocardiography (if age of donor gt 40)
Step 3
special investigations CT-scans abdomen and hepatic vasculature with volumetry MRI biliary system and Doppler
ultrasound of the carotid arteries (if age of donor gt 40)
screening of procoagulation disorders protein C protein S antithrombin III cardiolipin and anti-phospholipid
antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
The evaluation records of excluded donors
were retrospectively studied to classify causes
of late donor exclusion moreover the impact
of donor exclusion in changing the evaluation
protocol was addressed According to our
protocol of evaluation step 1-3 included the
main fundamental procedures for evaluation
however step 4 was constructed to include
final preoperative preparation measures
Donors were considered to be accepted for
donation when he she approved in steps 1-3
without any contraindication of donation but
who were not chosen to be donors for medical
andor psychological reasons during any of
the first three steps were considered excluded
In this study we considered and defined
patients to be late excluded (last minute
exclusion) if they were eliminated for any
reason during step 4 (of final preoperative
preparation) including after donor laparo-
tomy
Results
Of the 2500 potential living donor that had
been evaluated for suitability for liver
donation 375 (15) were accepted and only
194 (78) were donated Sixteen potential
donors were imposed to late donor exclusion
that caused by 8 different groups of cause
Exclusion reasons included late psychoso-
matic reasons in 4 donors family pressure to
withdraw consent in one donor and substance
abuse in 2 donors Early pregnancy was
discovered in one female donor week pre-
transplantation The pre-operatively revised
blood group of another donor was discovered
to be incompatible as the previously
determined blood group was incorrect The
presence of Factor V Leiden homozygous
mutation was diagnosed late in 2 donors In
four cases LDLT was aborted after donor
laparotomy due to macroscopic diffuse
hepatic changes considering the liver
unsuitable for donation Most of these
exclusions evoked discussions that led to a
sort of modifications in the evaluation
protocol and had an impact on both donors
and recipients One donor was convicted and
imprisoned 2 weeks before LDLT
Psychosomatic Reasons
We reported late psychosomatic reasons of
late donor exclusions in 4 donors The causes
were psychological instability with intense
psychological stress in two donors and
continuing hesitancy made another two
donors to withdraw their consent one day pre-
transplantation The former psychological
assessment carried a drawback of being
disorganized non-specialized and inefficient
So it was blamed to delay such exclusions
with resultant loss of the resources in
addition to its psychological impact for
recipients Subsequently early specialized
psychological assessment for all donors in
step 1 with stepwise protocol of psychoso-
matic assessment were added to our protocol
of donor evaluation
Social Problem
Donor family pressures prohibited a mother to
donate to her baby with liver failure
secondary to biliary atresia and they
prevented her from hospital admission three
days before the date of operation While
additional social problem in the form of
family opposition to the concept of donation
had been noticed it was under estimated and
donor evaluation proceeded till its end
Afterward early specialized psychological
assessment for all donors contributed to early
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
recognition of these pressures Moreover
Initiating of a family directed discussion
session to clarify justification of the LDLT
riskbenefit of the procedure and donor safety
led to a subsequent improvement in the family
acceptance of the concept of transplantation
with avoidance of such late exclusion
Substance Abuse
Donors with a history of previous occasional
drug abuse was not excluded and donated
without additional problems Two patients
denied drug abuse in history given on step 1
but they declared of ongoing addiction to
drugs in step 4 (opiates in one potential
donor and mixed drugs opiates and canna-
benoids in another one) and they were
excluded from donation for fear of associated
psychological instability and withdrawal
symptoms Accordingly after a short
discussion drug assay was added routinely to
step 2 of the evaluation protocol for all
potential donors
ABO-Incompatible Blood Group
According to our protocol potential donors
should have a blood group compatible with
that of the recipient (as our centre does not
accept incompatible transplantation) In one
case we faced a very unusual problem as the
reported ABO blood group of one donor
revealed to be incorrect and incompatible to
the recipient blood group during check of
blood matching day before operation after
being accepted and fully evaluated till blood
preparation for transplantation and that donor
was excluded This mistake was owed to the
dependence on small not credited laboratory
Although it is rare extraordinary mistake
from that time the decision had been taken to
do double check for ABO blood group in
credited laboratory for all cases
Donor Pregnancy
According to our protocol potential female
donor in child bearing period should stop
receiving oral combined contraceptive pill
(OCCP) 4-6 weeks pretransplantation due to
fear from hypercoagulable state with possible
serious thrombotic complications Early
pregnancy was discovered in one female
donor week pre-transplantation She was
taking OCCP that was stopped 6 weeks
before the scheduled date of transplantation
depending for contraception on safty periods
only Unfortunately she got pregnancy and
excluded from donation and this led to lose of
chance for transplantation as she was the only
suitable donor for her recipient As a result
double contraceptive measures to insure the
effectiveness of contraception became
recommend for any potential female donor
with stoppage of OCCP
Factor V Leiden Homozygous Mutation
As regard the initial protocol screening of
procoagulation disorders included protein C
protein S antithrombin III anti-cardiolipin
and anti-phospholipid antibodies If a liver
transplant donor or recipient has a personal
history of thrombosis a full pre transplant
hypercoagulable workup including Factor V
Leiden (FVL) mutation was done Later on
with availability of screening test for FVL
mutation and dating from 2009 (case number
70 and the subsequent cases) fully screening
of all procoagulation disorders including
FVL mutation became performed for all
donors as a routine in step 3 of evaluation
with exclusion of FVL homozygous
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
individuals from donation As a result the
presence of FVL homozygous mutation was
diagnosed late in 2 donors (as step 4 was
proceeded awaiting FVL result) so these two
donors were excluded late pretransplantation
In the later cases step 4 evaluation did not
start till the result of FVL to avoid such late
exclusion
Unexpected Finding During Donor
Laparotomy
Liver biopsy which is a mandatory part of the
evaluation performed routinely in step 2 of
our donor evaluation protocol this process
was performed under ultrasound guidance and
consisted of three core biopsies results of
10 or less fat infiltration were accepted
Four LDLT were aborted after donor laparo-
tomy due to macroscopic diffuse hepatic
changes considering the liver unsuitable for
donation Unpredicted findings at the time of
surgery included the presence of grossly
abnormal liver appearance with significant
gross hepatic fibrosis (figure 1) While these
donors were accepted for donation their
pathological reports showed mild periportal
infiltration in two cases presence of few
inflammatory cells of uncertain cause in one
case and presence of few unexplained
epitheloid granuloma in the last one These
exclusions after donor laparotomies evoked a
decision of double pathological revision of
the liver biopsy by two different expert
pathologists in the presence of any abnormal
finding even if very minimal Also laparo-
scopic assessment for debatable donors had
been suggested whenever liver biopsy results
for steatosis percentage did not give solid
satisfaction unexplained fibrosis or uncertain
finding with bizarre inflammatory cells
Fig 1 liver unsuitable for donation during donor laparotomy
Subsequently laparoscopic assessment had
been performed in step 2 of evaluation for
24 donors with debatable findings using two
ports of 3 and 5 mm during which gross
evaluation of the donor liver was performed
in addition large wedge biopsies were taken
for confirmation Depending on the results of
laparoscopic assessment 9 donors were
excluded early in stage 2 while the other 15
donors were accepted and the evaluation
proceeded of whom eight donors had been
finally donated
Donor Imprisoning
Unfortunately one donor was convicted and
imprisoned week before LDLT and this led to
his exclusion Several changes that had been
imposed to our initial protocol were
illustrated in table 2
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Step 1
Clinical evaluation Medical history and physical examination
relation to recipient age weight and size medical history ABO compatibility
Laboratory tests Erythrocyte sedimentation rate differential blood count electrolytes liver kidney and
pancreas profile glucose coagulation profile urine analysis Serology for HBV HCV HIV CMV EBV
HSV
Abdominal ultrasound
Initial expert psychological and ethical evaluation
Step 2
Liver biopsy double assessment
Laparoscopic assessment in uncertain cases
Cardiopulmonary evaluation Electrocardiography Stress electrocardiography Chest radiography
Pulmonary function test Echocardiography (if age of donor gt 40)
Drugs assay
Step 3
Special investigationsCT-scans abdomen and hepatic vasculature with volumetry MRI biliary system
and Doppler ultrasound of the carotid arteries (if age of donor gt 40)
Detailed screening of procoagulation disorders protein C protein S antithrombin III factors V Leiden
mutation prothrombin mutation homocystein factor VIII cardiolipin and anti-phospholipid antibodies
Step 4
Final medical evaluation
Preoperative anesthesia evaluation and consent of the donor
Planning of the surgical date and availability of ICU facilities
blood preparation and autologous blood donation
Final psychological and ethical evaluation
Table 2 final protocol after modifications
Discussion
Donor safety has always been a major
concern and potential risk to the donor must
be balanced against recipient benefit
However lack of a standardized and uniform
evaluation of perioperative complications is a
serious limitation of the evaluation of donor
morbidity(8) Top priority must be given to
developing guidelines for effective donor
selection for each medical discipline
involved(9) only 89 (14) of the first 622
donors for adult recipients were considered
suitable for donation after the evaluation
potential donors for living-donor liver trans-
plantation in a single center(1) Moreover
Emond et al 1996(5) and Chen et al 1996(6)
reported that acceptance rate for donors
undergoing the evaluation process vary
between 22 and 66 Also Trotter et al
2002(7) reported that only 49 of 100 potential
recipients were found to be suitable for
LDLT Of these only 25 had a potential
donor of which 15 were finally accepted
Similar to these results of the 2500 person
that had been evaluated for suitability for liver
donation in our centre only 15 were
accepted for donation and 194 (78) were
finally donated Psychosocial assessment is
one of the most important steps in donor
evaluation(10) Psychosocial problems are one
of the most frequent reasons for excluding
donor candidates for LDLT(11) It is important
that patients and their families feel no
pressure in seeking an appropriate person for
donation The ideal situation is one in which
the potential donor spontaneously presents
himself for evaluation(12 13) Six candidates
were rejected after the psychosomatic
interview because of family coercion in the
report by Erim et al 2008(10) In our centre
we faced family pressures against
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
transplantation fearing from operative
outcome At that time this was explained by
the culture of our community which was
relatively against the concept organ donation
Under estimation of this social problem in
addition the inefficient psychological assess-
ment led to this case of late exclusion
Specialized psychological assessment contri-
buted to early recognition of these pressures
with avoidance of such late exclusion Donors
should never be compelled to donate The
psychological evaluation focuses on the
emotional stability of the potential donor and
is used to verify the informed consent Donors
should be permitted to change their mind up
until the induction of general anesthesia and
they should be given every opportunity to
withdraw at any time if they have any fears
Psychological counseling can be very helpful
and multiple consents encourage donors to
reconsider their decision(14) Valentin-Gamazo
2004(1) presented 5 steps of course of the
psychosomatic assessment protocol for
potential living liver donors Also results of
Erim et al 2008(12) indicated that candidates
with mental disturbances and additional social
distress ambivalent candidates and those in
difficult family relationship were excluded in
the psychosomatic evaluation accordingly
eleven donor candidates were rejected
because of mental vulnerability and three
donor candidates were excluded because of
indecisiveness In our centre donors
permitted to withdraw at any time if they have
any fears Psychological instability and
continuing hesitancy caused late exclusion in
4 donors pre-transplantation The drawback
was the dependence on disorganized non-
specialized and inefficient psychological
assessment There were discussions about
whether these donors might be excluded early
with saving of resources if proper
psychosomatic assessment was done Sub-
sequently early specialized psychological
assessment for all donors in step 1 with
stepwise protocol of psychosomatic asse-
ssment were added to our protocol of donor
evaluation Erim et al 2008(12) excluded six
patients had a diagnosis of ongoing addiction
to alcohol or other drugs In our centre two
potential donors with ongoing substance
abuse were excluded and this was justified by
the inability to expect severity of post-
operative withdrawal symptoms and to
estimate the probable increase in post-
operative risk Moreover the probability of
worse outcome if donor hepatectomy per-
formed for individual with substance abuse
did not studied before Accordingly drug
assay were added to step 2 of the evaluation
protocol for all donors aiming to detection of
donors with drug abuse with early exclusion
of them Potential donors should have a blood
group compatible with that of the recipient
ABO - incompatible donors have been
described but should remain exceptional(15 16)
we do not accept incompatible transplantation
in NLI Surprisingly blood group of one
donor was discovered to be incompatible
during preoperative blood matching as the
previously determined blood group was found
to be incorrect and this donor was excluded
Although it is a very rare and unusual error
from that time the decision had been taken to
do double check for ABO blood group in
credited lab as a protocol in all cases Post-
operative complications in live donors
especially during the first few months include
pulmonary embolism with an incidence of
7 so of which were fatal(17) Deep venous
thrombosis spleen and portal vein thrombosis
have been reported as part of the serious
thrombotic complications Overall there is
underestimation and under-appreciation of the
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
thromboembolic risks in the living donors(17)
Living donors progressively developing
hypercoagulable state even in the presence of
anti-thrombotic prophylaxis(18) Hypercoagul-
able states are relative contraindications for
donation for fear of increase donor mortality
from pulmonary embolism(19) Several large
studies have confirmed a two-fold to six-fold
increased risk of deep venous thrombosis
associated with current oral contraceptive
use(20-23) later on Tan and Marcos 2004(24)
recommended cessation of oral contraceptives
4-6 weeks prior to LDLT in summarizing the
ideal LDLT donor profile This was also
adopted in our protocol for all women donors
in child bearing period however early
pregnancy was discovered in one female
donor week pre-transplantation She was
receiving OCCP that was stopped 6 weeks
preoperative and she depended for contra-
ception on safety periods only Unfortunately
she got pregnant and excluded from donation
and this led to lose of the chance of
transplantation for this recipient as she was
the only suitable donor Therefore double
contraceptive measures to insure the
effectiveness of contraception became recom-
mend for potential female donors with
stoppage of OCCP Up to 50 of patients
with hepatic artery thrombosis (HAT) may
require retransplantation(25) A thrombotic
event in the liver graft all too often results in
prolonged hospitalization graft loss retrains-
plantation or patient death In fact about
16 of all liver allograft failures were
secondary to thrombotic events These events
are associated with an increased use of
resources and costs(26-28) The Factor V Leiden
(FVL) mutation is the most common genetic
defect predisposing to venous thrombosis(29)
FVL heterozygosity increases the life time
risk of venous thrombosis by 5- to 10-fold
and 50- to 80 - fold in homozygous
individuals (30) The FVL mutation causes
factor Va to be resistant to cleavage by
activated protein C (APC) resulting in more
factor Va being available thereby increasing
the generation of thrombin Thus the FVL
mutation and the resultant APC resistance
cause a hypercoagulable state (31 32)
Identifying APC resistance in liver donors
could allow the clinician to expectantly care
for liver recipients according to known risk
factors and should decrease hepatic vascular
thromboses As we strive toward decreasing
morbidity and cost testing for APC resistance
will further improve outcome parameters(33)
On the other hand Gideon et al 1998(34)
reported that the factor V Leiden mutation in
the donor liver is not a major risk factor for
hepatic vessel thrombosis and subsequent
graft loss after liver transplantation Also
Brian 2004(35) stated that venous thrombosis
does not appear to be increased in the
presence of FVL heterozygosity and there is
no evidence for altering perioperative
management on the basis of the finding of
FVL nor is there evidence to support a role of
preoperative screening for FVL or a PC
resistance In the earlier era of our LDLT
program in NLI screening for FVL mutation
was performed in very limited number of
laboratories with high cost and long duration
So it was performed in selected high risk
cases Gradually this test become more
available with relatively less cost and
duration With the occurrence of not fully
explained thrombotic complications (which
led to graft loss) in some cases the awareness
about increased risk of thrombotic events
associated to FVL mutation became more
obvious Previously Dieter et al 2003(36)
stated that It is a matter of debate whether
potential donors with a mildly increased risk
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
for thrombotic events as in heterozygote
carriers of a factor V Leiden gene mutation
should be excluded from donation This
debate was stopped from 2009 in our centre
by making a decision that only FVL
homozygous individuals should be excluded
from donation but the presence of FVL
heterozygosity is accepted for donation with
precautions and that step 4 of preparation
should not start awaiting the result of FVL to
avoid late donor exclusion Liver biopsy is a
routine step in donor evaluation in a high
percentage of LDLT programs Other
methods to evaluate the fat content of the
donorrsquos liver are less sensitive and specific
than liver biopsy and these alternatives are
unable to detect any associated liver
pathology Unfortunately liver biopsy is an
invasive technique and is associated with a
certain risk of complications Recent studies
have reported an incidence of major
complications related to liver biopsy of 13
(37-39) In our centre liver biopsy which is a
mandatory part of donors evaluation was
performed routinely in step 2 of our donor
evaluation protocol There have been reports
of aborted donor hepatectomy at the time of
surgery as a result of unexpected findings
including the presence of significant hepatic
steatosis(40) We reported discontinuation of
LDLT In four cases after donor laparotomy
due to macroscopic diffuse hepatic changes
considering the liver unsuitable for donation
Subsequently any argument about the result
of liver biopsy was solved by re-evaluation by
two different expert pathologists otherwise
laparoscopic assessment for the potential
donor liver is performed The aim was to
early alleviate the controversy around the
suitability for donation and to avoid donor
exclusion after laparotomy According to
Hegab et al 2012(41) 30 potential living
donors were assessed laparoscopically in the
day of surgery to determine whether to
proceed with the abdominal incision to
harvest part of the liver for donation and they
concluded that the laparoscopic assessment of
donors in LDLT is a safe and acceptable
procedure that avoids unnecessary large
abdominal incisions and increases the chance
of achieving donor safety In our centre
laparoscopic assessment had been performed
in step 2 of evaluation for 24 donors with
debatable findings and helped to early fading
away of these debates Our observations about
late donor exclusion indicated that it affected
recipients resources and evaluation protocol
Late exclusion carried psychological effect
for recipient and family also it increased
transplantation cost and led to lose of more
resources Moreover the important issue is
the impact of these exclusions in the
evaluation protocol as each of them evoked
discussions that led to a kind of modification
in the steps of the initial evaluation protocol
Conclusion
Late pre-transplantation donor exclusion had
its impact in donors recipients and resources
Early expert well organized psychological
assessment is crucial and should be a part of
any evaluation protocol Laparoscopic
assessment of donors in LDLT is a safe and
acceptable procedure that avoids late
operative exclusions and it is useful in
debatable cases Reassessment of the donor
evaluation protocol should be a dynamic
process and it found to be greatly affected by
these late exclusions
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
References
1 Valentiacuten-Gamazo C Malagoacute M Karliova M et al
Experience after the evaluation of 700 potential
donors for living donor liver transplantation in a
single center Liver Transpl 2004 10 1087-1096
2 Pomfret EA Early and late complications in the
right-lobe adult living donor Liver Transpl 2003
9 S45-S49
3 Broering DC Sterneck M Rogiers X Living
donor liver transplantation Journal of Hepatology
2003 38(S)119ndashS135
4 Henkie PT Kusum PT and Amadeo M Adult
living donor liver transplantation Who is the ideal
donor and recipient 2005 (43) 1 13-17
5 Emond JC Renz JF Ferrell LD et al Functional
analysis of grafts from living donorsImplications
for the treatment of older recipientsAnn
Surg1996224544ndash552
6 Chen YS Chen CL Liu PP et al Preoperative
evaluation of donors for living related liver
transplantation Transplant Proc1996 282415ndash
2416
7 Trotter JF Wachs M Everson GT et al Adult-to-
adult transplantation of the right hepatic lobe from
a living donorN Engl J Med 2002 346 (14)1074ndash
82
8 Ding Y Yong-Gang W Bo L et al Evaluation
outcomes of donors in living donor liver
transplantation a single-center analysis of 132
donors Hepatobiliary amp Pancreatic Diseases
International Volume 10 Issue 5 October 2011
Pages 480ndash48
9 Erim Y Malago M Valentin-Gamazo C et al
Guidelines for the psychosomatic evaluation of
living liver donors analysis of donor exclusion
Transplant Proc 2003 35909ndash910
10 Erim Y Beckmann M Valentin-Gamazo C et al
Selection of donors for adult living-donor liver
donation results of the assessment of the first 205
donor candidates psychosomatics 2008 49143ndash
151
11 Sterneck MR Fischer L Nischwitz U et al
Selection of the living liver donor Transplantation
1995 60667ndash671
12 Schiano TD Kim-SchlugerL GondolesiG et
al Adult living donor liver transplantation the
hepatologists perspectiveHepatology 33 (2001)
pp 3ndash9
13 Pszenny C Krawczyk M Paluszkiewicz R et al
Biochemical function of the donor liver in living
related liver transplantation Transplant Proc
200234621ndash622
14 Marcos A Fisher R Ham J et al Selection and
outcome of living donors for right lobe liver
transplantation Transplantation2000
Jun1569(11)2410-5
15 Rydberg L ABO-incompatibility in solid organ
transplantation Transfus Med 200111325ndash342
16 Tanabe M Shimazu M Wakabayashi G et al
Intraportal infusion therapy as a novel approachto
adult ABO- incompatible liver transplantation
Transplantation 2002731959ndash1961
17 Bezeaud A Denninger MH Dondero F et al
Hypercoagulability after partial liver
resection Thromb Haemost 2007 981252-1256
18 Cerutti E Stratta C Romagnoli R et al
Thromboelastogram monitoring in the
perioperative period of hepatectomy for adult
living liver donation Liver Transpl 200410289-
294
19 Durand F Ettorre GM Douard R et al Donor
safety in living related liver transplantation
underestimation of the risks for deep vein
thrombosis and pulmonary embolism Liver
Transpl 20028118ndash120
20 Vandenbroucke JP Koster T Brieumlt E et al
Increased risk of venous thrombosis in
oralcontraceptive users who are carriers of factor
V Leiden mutation Lancet 19943441453-7
21 Thorogood M Mann J Murphy M et al Risk
factors for fatal venous thromboembolism in
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
young women a case-control study Int J
Epidemiol 19922148-52
22 WHO Venous thromboembolic disease and
combined oral contraceptives results of
international multicentre case-controlstudy World
Health Organization Collaborative Study of
Cardiovascular Disease and Steroid Hormone
Contraception Lancet 19953461575-82
23 Farmer RD Lawrenson RA Thompson CR et al
Population-based study of risk of venous
thromboembolism associated with various oral
contraceptives Lancet 199734983-8
24 Tan HP Marcos A Hepatic arterial anatomy for
right liver procurement from living donors Liver
Transpl 2004 10 134ndash135
25 Settmacher U Stange B Haase R et al Arterial
complications after liver transplantation Transpl
Int 2000 13 372
26 Taylor MC Greig PD Detsky AS et al Factors
associated with the high cost of liver
transplantation in adults Can J Surg 2002 45
425
27 Brown RS Jr Ascher NL Lake JR et al The
impact of surgical complications after liver
transplantation on resource utilization Arch Surg
1997 132 1098
28 Whiting JF Martin J Zavala E et al The
influence of clinical variables on hospital costs
after orthotopic liver transplantation Surgery
1999 125 217
29 Rees DC Cox M Clegg JB World distribution of
factor V Leiden Lancet 1995 346 1133
30 Bauer KA Rosendaal FR Heit JA
Hypercoagulability too many tests too much
conflicting data Hematology (Am Soc Hematol
Educ Program) 2002 353
31 Bertina RM Koeleman BP Koster T et al
Mutation in blood coagulation factor V associated
with resistance to activated protein C Nature
1994 369 64
32 Zoller B Hillarp A Berntorp E et al Activated
protein C resistance due to a common factor V
gene mutation is a major risk factor for venous
thrombosis Annu Rev Med 1997 48 45
33 Christella M Thomassen LG Castoldi E et al
Endogenous factor V synthesis in megakaryocytes
contributes negligibly to the platelet factor V pool
Haematologica 2003 88 1150
34 Gideon H Jane DC Karen B et al Donor Factor
V Leiden Mutation and Vascular Thrombosis
Following Liver Transplantation Liver
Transplantation and Surgery Vol 4 No 1 (
January) 1998 pp 58-61
35 Brian SD Factor V Leiden and Perioperative Risk
Anesth Analg 2004 981623ndash34
36 Dieter CB Martina S Xavier R Living donor
liver transplantationJournal of Hepatology 2003
38 S119ndashS135
37 Rinella ME Abecassis MM Liver biopsy in living
donors Liver Transpl 2002 8 1123-1125
38 Brandhagen D Fidler J Rosen C Evaluation of
the donor liver for living donor liver
transplantation Liver Transpl 2003 9 S16-S28
39 Nadalin S Malagoacute M Valentin-Gamazo C et al
Preoperative donor liver biopsy for adult living
donor liver transplantation risks and benefits
Liver Transpl 2005 11 980-986
40 El-Meteini M Fayez M Abdalaal A et al Living
related liver transplantation in Egypt an emerging
program Transplant Proc 2003 35(7)2783-2786
41 Hegab B Abdelfattah M R Azzam A et al Day-
of-surgery rejection of donors in living donor liver
transplantation World J Hepatol 2012 November
27 4(11) 299-304
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Original Article
The Impact of Interleukin 28B Tumor Necrosis Factor Alpha Gene
Polymorphisms on Therapeutic Response in Chronic HCV Egyptian Patients
Hamouda S Hanno A El-Shayeb A Mansour A Elwazzan D
Department of Tropical Medicine Department of Clinical Pathologyy Alexandria UniversityEgypt
ABSTRACT
Chronic chronic hepatitis C virus (HCV) infection is a major health problem in Egypt The currently recommended
therapy of HCV infection is the combination of a pegylated interferon (PEG-IFN) alfa and ribavirin therefore reliable
markers that predict the response to therapy is of great importance from the economic point of view Objectives The
aim of this work was to find the effect of polymorphism of Interleukin 28B (IL28B) and Tumor Necrosis Factor alpha
(TNF-a) genes on the response to combination therapy (PEG-IFN and ribavirin) in chronic HCV infected Egyptian
patients Patients and methods 150 subjects divided into two groups group (I) one hundred patients with chronic
HCV who were candidates to receive combination therapy with interferon ribavirin they were subjected to all
investigations needed before IFN therapy in addition to IL28B 12979860 (using Conventional ARMS-PCR) and TNF-a
308 (using the allele specific primer conventional PCR)genotyping then received combination therapy with estimation
of HCV RNA at weeks 12 24 and six months after the end of therapy Group (II) fifty healthy subjects as a control
group also were subjected to IL28B 12979860 and TNF-a 308 genotyping Results IL28B rs12979860 and TNF-a
rs308 genotypes were observed to have a statistically significant association with the response to treatment in chronic
HCV (CHC) patients (p=000 and 0034 respectively) The IL28B C allele was higher in the responders while the T
allele was higher among the non-responders TNF-a G allele was significantly higher among the responders while the
A allele was significantly higher among the non-responders Conclusion IL28B rs12979860 and TNF-a rs308
genotyping can be used as predictors of response to combination therapy in CHC Egyptian patients
Introduction
The estimated global prevalence of HCV
infection is 3 corresponding to about 130-
210 milion HCV-positive persons worldwide
the highest prevalence (15-22) has been
reported from Egypt (12) HCV-infected people
serve as a reservoir for transmission to others
and are at risk for developing chronic liver
disease cirrhosis and primary hepatocellular
carcinoma (HCC) (3) The treatment of
hepatitis C has evolved over the years
Current treatment is combination therapy
consisting of ribavirin and IFN to which
polyethylene glycol (PEG) molecules have
been added (ie PEG-IFN) that increased the
sustained virological response (SVR
undetectability of HCV RNA 6 months after
stopping treatment) rate almost 10 fold up to
54ndash61(4) but unfortunately this combination
regimen has a number of drawbacks
Intolerable side effects necessitate prema-
turely stopping treatment in about 15 of
patients and dose reductions in another 20ndash
40 Moreover the drug regimen is very
expensive which means that most patients in
countries such as Egypt which has 10ndash12
million infected individuals cannot afford
this therapy (5) Accordingly identification of
the predictors of response to treatment is a
high priority(6) A number of viral and host
factors associated with response to interferon-
based therapy have been identified Viral
factors are limited to viral genotype HCV
RNA titer and possibly mutations in certain
regions of the viral genome In addition
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
several host factors are associated with
response such as patient age sex ethnicity
and cytokine polymorphisms Among these
cytokines IL28B and TNF-a (7) Genome
wide association studies (GWAS) have
identified single nucleotide polymorphisms
(SNP) near the IL28B gene Favorable variant
of the most widely studied IL28B
polymorphism rs12979860 is strongly
associated with both spontaneous and
treatment - induced viral clearance (8 9) The
IL28B genotype greatly influences response
to IFN-based therapy and so is most relevant
in the less interferon - responsive viral
genotypes (ie genotypes 1 and 4) (10) The
actual causal polymorphism at this locus
remains unknown Some studies suggested
that their IL-28B polymorphism was
associated with decreased IL-28B (and IL-
28A) expression that encodes interferon-
lambda3 (IFNλ) which may be involved in
interfering with viral replication (11 12)
Results in three possible genotypes revealed
that SVR in patients with the CC genotype is
greater than that seen in patients with a CT
genotype which is in turn greater than that
seen in patients with a TT genotype The
rs12979860 polymorphism may also explain
much of the difference in response between
different population groups in fact the C
allele which leads to better response presents
with greater frequency in European than in
African populations(13) TNF-a a potent
antiviral proinflammatory cytokine that
encoded by major histocompatibility complex
class III region on chromosome 6p21 has
been implicated as an important pathogenic
mediator in a variety of liver conditions (14)
TNF-a plays a pivotal role in host immune
response to HCV infection because cytotoxic
T lymphocytes in the liver secrete TNF-a(15)
Circulating TNF-a level increases during
HCV infection that induces TNF-a production
in human hepatocytes(16) TNF-a expression is
tightly controlled at the transcriptional and
posttranscriptional levels(17) The maximal
capacity of cytokine production varies
between individuals and may correlate with
polymorphism in cytokine gene Many
diallelic polymorphisms in the TNF-a which
are thought to affect TNF-a production have
been reported the most important is the GA
transition at position 308 (18 19) which is
thought top play a role in the pathogenesis of
acute and chronic HCV infection the
persistence of the virus (214)and the response
to IFN therapy (14)
Subjects and Methods
This study included 150 subjects divided into
two groups group (I) one hundred patients
with chronic HCV who were treatment naiumlve
for hepatitis C and appropriate candidates to
receive combination therapy with interferon
ribavirin They were collected from the
Tropical Medicine Outpatient Clinic-
Alexandria University group (II) fifty
healthy subjects of matched age and sex as a
control group An informed consent was
signed by every subject prior to start of the
research Blood samples were collected from
all patients to assess the following
parameters routine laboratory studies
complete liver profile and viral markers
including HBsAg assay HCV Ab and
quantitative HCV RNA Abdominal
ultrasonography ultrasound guided percutanous
needle liver biopsy were also done for all group
(I) patients using a Trucut liver biopsy needle
Grading and staging of chronic hepatitis were
evaluated hisologically according to the
intensity of necroinflammatory changes and
fibrosis respectively using METAVIR scoring
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
system All patients in group (I) received
combination therapy (pegylated IFN once
weekly injection plus ribavirin oral 1000-1200
mg per day according to body weight)
Quantitative HCV RNA was done at weeks
12 24 and 6 months after the completion of
treatment if the HCV RNA was undetectable
or if a greater than 2-log-fold reduction in
HCV RNA level was present in week 12 the
patient was considered as a responder and the
therapy was continued by the same regimen
Conversely if response was not achieved less
than 2-log-fold reduction in HCV RNA level
than baseline at week 12 or reappearance of
HCV RNA in serum while still on therapy or
after therapy is discontinued the patient was
considered as non responder and the treatment
was discontinued IL28B rs12979860 and
TNF-a rs308 genotyping Genomic DNA was
extracted from a 2 ml blood sample then
IL28B rs12979860 genotyping was done by
using a conventional ARMS-PCR (21)while
the TNF-a 308 GA SNP was determined
using the allele specific primer conventional
PCR (22)
Statistical Analysis
Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc) Data
were presented as means with corresponding
standard error (SE) Comparisons among
different groups were performed by one way
analysis of variance (ANOVA) Qualitative
data were described using number and percent
and association between categorical variables
was tested using Chi-square test Correlation
between variables was determined using
Pearsons or Spearmans correlation test
according to the variable In all tests the level
of significance was lt 005
Results
HCV RNA follow up According to the
results of HCV RNA (quantitative PCR)
during the course of treatment and six months
after the end of therapy those who achieved
SVR were 67 (67) patient while the non-
responders (whether during or after the end of
treatment) were 33(33) patients
Distribution of IL28B varients In group I 38
(38) patients carrying the CC genotype CT
genotype present in 48 (48) patients while
only 14 (14) patients having TT genotype
Subsequently the C allele was found in 62
of patients vs 38 for the T allele In group
II CC genotype was found in 28 (56) CT
genotype present in 18 (36) individuals
while 4 (8) individuals carrying the TT
genotype This means that the C allele present
in 74 vs 26 T allele of individuals in
group IV (table I amp figure 1)
Table (I) IL28B genotypes among different groups
CC No () CT No () TT No () CT ratio
Group I 38 (38) 48 (48) 14 (14) 6238
Group II 28 (56) 18 (36) 4 (8) 7426
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Figure (1) Frequencies of IL28B genotypes as of total number of subjects in each group
Correlation between IL28B genotypes and response to treatment in group I patients
Among the responders to treatment (67
patients) the CC genotype was found in 34
(507) patients the CT genotype present in
31 (463) patients while TT genotype was
detected only in 2 (3) patients Within the
non-responders (33patients) the CC genotype
was found in 4 (121) patients the CT
genotype present in 17 (515) patients
while TT genotype was detected in 12
(364) patients IL28B genotypes were
observed to have a statistically significant
association with the response to treatment in
group I patients (The C allele was higher in
the responders while the T allele was higher
among the non-responders) Chi-square
(X2=264) (p=000) (Table II amp figure 2)
Table (II) IL28B genotypes among the responders and non-responders
IL28B genotypes
CC No () CT No () TT No () CT
ratio
Total
(NO)
Response to
therapy
Responders
34 (507) 31 (463) 2 (3) 7426 67
Non-responders
4 (121) 17 (511) 12(364) 3862 33
Total (n) 38 48 14 100
X2 264
P 000
Significant at P 005
Figure (2) Correlation between IL28 genotypes and response to treatment in group I patients
0
10
20
30
40
50
60
Group I Group II
CC
CT
TT
0
20
40
60
Responders Nonresponders
CC
CT
TT
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
TNF-a genotypes in different groups In
group I the GG genotype was found in 79
patients (79) while GA genotype was found
in 21 patients (21) This means that the G
allele present in 895 of patients vs 105
for the A allele In group II GG genotype was
found in 14 (56) patients while GA
genotype present in 11 (44) patients So the
G allele was found in 78 of patients vs 22
for the A allele
Table (III) TNF-a genotypes among different groups
GG No () GA No () GA ratio
Group I 79 (79) 21 (21) 895105
Group II 46 (92) 4 (8) 964
Figure (3) Frequencies of TNF-a genotypes as of total number of subjects in each group
Correlation between TNF-a genotypes and
response to treatment in group I patients
tables (IV) amp figure (4) Among the
responders to treatment the GG genotype was
found in 57 (851) patients while the GA
genotype was detected in 10 (149) patients
Thus the G allele was found in 925 and A
allele present in 745 of patients Within the
non-responders the GG genotype was found
in 22(667) patients while GA genotype
was detected in 11 (333) patients So the G
allele was found in 834 while the A allele
was detected in 166 of patients TNF-a
genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients Chi-
square (X2=451) (p=0034) The G allele was
significantly higher among the responders
while the A allele was significantly higher
among the non-responders
Table (IV) TNF-a genotypes among the responders and non-responders
TNF-a genotypes
GG No () GA No () GA ratio Total (n)
Response to therapy Responders 57 (851) 10 (149) 925 745 67
Non-responders 22 (667) 11(333) 834 166 33
Total (n) 79 21 100
X2 451
P 0034
Significant at P 005
0
20
40
60
80
100
Group I Group II
GG
GA
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
Figure (IV) Correlation between TNF-a genotypes and response to therapy
IL28B CC and CT showed PPV of 849 and
645 respectively in predicting the response
to treatment while the TT genotype showed
857 PPV in predicting the non response to
therapy Moreover the CC and CT showed
efficacy of 63 and 47 respectively in
predicting the response to treatment while the
TT genotype showed 77 efficacy in
predicting the non response to therapy
Regarding TNF-a the GG genotype showed
721 PPV in predicting the response to
treatment while the GA genotype showed
523 PPV in predicting the non response to
therapy furthermore both GG and GA
genotypes showed an efficacy of 68 in
predicting the response and non response to
treatment The PPV and efficacy of combined
IL28B CC genotype and TNF-a GG genotype
were 968 and 75 respectively while the
PPV and efficacy of combined IL28B TT
genotype and TNF-a GA genotype were 56
and 45 respectively
Table (V) PPV and efficacy of IL28B and TNF-a
IL28B TNF-a Combined IL28B and TNF-a
CC CT TT GG GA CC and GG TT and GA
PPV 894 645 857 721 523 968 75
Efficacy 63 47 77 68 56 45
Discussion
It is now proved that IL28B rs12979860
genotypes are strongly associated with HCV
infection outcomes spontaneous and
treatment induced viral clearance (8-10) Many
studies suggested that IL-28B polymorphism
is associated with decreased IL-28B (and IL-
28A) expression that encodes IFNλ 3 which
may be involved in interfering with viral
replication (11 12) In many studies it was
found that the favorable C allele of IL28B
rs12979860 was found to be more frequent in
Asian ethnicity than Caucasian and African
populations respectively(23) In the present
work it was observed that IL-28B
rs12979860 CC genotype frequency
decreased from 56 among healthy subjects
0
20
40
60
80
100
Responders Nonrespondres
GG
GA
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
vs 38 among CHC patients regardless of
their response to combination therapy
Furthermore the frequency of C allele was
62 in CHC patients vs 74 in healthy
subjects A comparable increase in the TT
genotype was observed in chronic HCV
patients vs healthy subjects (14 vs 8)
Regarding the heterozygote CT genotype it
was higher in chronic HCV patients compared
with the healthy subjects (48 vs 36) The
current results were in consistence with that
of El-Awady et al (24) that conducted a
research on HCV genotype 4 infected
Egyptian patients they found that there is
sharp decrease in the incidence of the
protective CC genotype and C allele
compared with the typing data of healthy
subjects 13 CC in chronic HCV patients
vs 48 CC in healthy subjects and 50 C
allele in CHC vs 67 C allele in healthy
subjects However such a decline in the CC
genotype was not reflected in a comparable
increase in the TT genotype (13 vs 14)
A two fold increase (75 vs 38) in the
heterozygous genotype CT was observed in
the CHC group compared with the healthy
subjects In a Korean study done in HCV
genotypes 1b and 2 infected patients the
prevalence of rs12979860 in healthy controls
was as follows the CC genotype was 885
the CT genotype was 115 and the TT
genotype was not found while in chronic
hepatitis C patients there was significant
decrease in the C allele and significant
increase in the T allele than that in healthy
control (25) As regards the response to
therapy to date a broad association between
favorable IL28B genotypes and SVR has been
described in patients infected with HCV
genotype 1 with a similar association
described for genotype 4 (26) although this has
been less studied However conflicting
results have been published about HCV
genotypes 23 (27) The generally reduced
association for patients with HCV genotypes
23 could be related to the high rate of SVR
present in these IFN-sensitive genotypes for
which larger sample sizes are required to find
significant differences(28) In the present
work it was noticed that IL-28B rs12979860
CC genotype incidence among the responders
and non-responders was 507 vs 121
respectively with a PPV of 849 in
predicting the response to treatment while the
reverse occurred for the TT genotype which
affected only 3 of the responders and 364
of the non-responders with a PPV of 857 in
predicting the non-response to therapy which
highlights the protective effect of the C allele
IL28B genotypes were observed to have a
statistically significant association with the
response to treatment in group I patients The
C allele was higher in the responders while
the T allele was higher among the non-
responders (X2=264) (p=000) The results of
the present work were in agreement with that
of Derbala et al(29) who did a similar research
in Egypt on HCV genotype 4 they found that
the patients who did not achieve ETR (End of
therapy response) or SVR had a lower
prevalence of rs12979860 CC (174 and
233 respectively) than individuals who had
ETR or SVR (479 and 472
respectively) Moreover they observed that
carrying at least one copy of the C allele
(genotypes CT and CC) had almost 8 times
the probability of ETR compared to those
with genotype rs12979860 TT concluding
that in HCV genotype 4 patients rs12979860
is a sensitive predictor of treatment induced
viral clearance Similarly in another research
done in Egypt by Pasha HF et al (30) they
noticed that the inheritance of IL28B CT and
TT genotypes may be associated with
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
susceptibility to HCV infection and resistance
to combined antiviral therapy Mangia A et al (27) detected different results they claimed
that IL28B type can not be used as a predictor
of SVR in HCV genotype I infected patients
and it appeared to have limited potential for
response-guided treatment strategies In
addition Lin CY et al(17) were in agreement
with Mangia et al (27) as they found that HCV
genotype 1 infected patients younger than 40
years old had SVR rate similar to HCV
genotype 1 infected patients and the IL28B
polymorphism had no impact on the SVR rate
in these patients In the current results as
regards TNF-a gene polymorphism it was
observed that in group I patients the GG
genotype frequency was higher than GA
genotype frequency the AA genotype was
absent In comparison to control group the
GG genotype was increased from 79 in
group I patients to 92 in control group
while GA genotype was decreased from 21
in group I patients to 8 in controls The
TNF-a typing profile in group I patients
displayed a clear inversion of G to A ratios to
become 895 105 instead of 96 4
found in healthy subjects Regarding the
response to treatment among those who
achieved SVR the GG genotype frequency
was significantly higher than the GA
genotype (851 and149 respectively)
while the reverse was detected among the non
responders (the GG genotype was found in
667 and the GA genotype was detected in
333) Thus the GG genotype was found to
be correlated to the response to combination
therapy with a PPV and efficacy of 721 and
68 respectively in predicting the response to
treatment while the GA genotype showed
lower PPV 523 PPV in predicting the non
response to therapy (523) The present
results were in agreement with that of Dai CH
et al (14) who demonstrated that the
distribution of TNF-a 308 promoter genotypes
in HCV genotype 1 infected patients were as
follows TNF308 GG was found in 766
TNF308 GA was found in 213 and
TNF308 AA was found in 21 of their
patients and the frequencies of the TNF308 G
allele and the TNF308 A allele were 872
and 128 respectively In the same study
after undergoing combination therapy they
found that significantly higher proportion of
TNF308 A allele non-carriers than carriers
had an SVR (981 vs 879 p=027) In
addition the carriers of the G allele were
found to have a lower fibrosis score and a
higher necro-inflammatory activity score than
did those who were A allele carriers which
was in agreement with the current results the
stage of liver fibrosis was observed to have a
statistically significant association with TNF-
a genotypes but the grade of necro-
inflammation was not associated with TNF-a
genotypes Pasha HF et al(30) documented
that the inheritance of TNF-α GA and AA
genotypes which appear to affect the cytokine
production may be associated with suscep-
tibility to HCV genotype 4 infection and
resistance to combined antiviral therapy In
disagreement to the current results Yee et
al(31) reported that there was no correlation
between TNF-a 308 polymorphisms and the
response to combination therapy in patients
with chronic HCV genotype 1infection
Moreover Abbas Z et al(31) demonstrated that
no significant differences in the degree of
necro-inflammatory activity and fibrosis were
noted among the TNF-a genotypes and they
did not modulate the response to combination
therapy for genotype 3infected patients they
attribute this to the fact that viral genotype 3
is easy to treat genotype in any case In the
current study it was found that combined
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
IL28B rs 12979860 CC genotype and TNF-a
rs 308 GG genotype had higher PPV and
efficacy in predicting the response to
combination therapy than testing for either
alone suggesting that testing for multiple
factors which influence the response to
treatment may be more helpful for the
physician in taking the decision for treatment
Conclusion
IL28B rs12979860 C allele and TNF-a rs308
G allele are significantly associated with
response to therapy while their T and A
alleles were found to be associated with non-
response therefore they can be used as
predictors of response to combination therapy
in CHC Egyptian patients
References
1 Alter MJ Epidemiology of hepatitis C virus
infection World Journal of Gastroenterology
2007 13 2436ndash41
2 Frank C Mohamed MK Strickland GT et al The
role of parenteral antischistosomal therapy in the
spread of hepatitis C virus in Egypt Lancet 2000
355 887-91
3 Lavanchy D The global burden of hepatitis C
Liver Int 2009 1 74-81
4 Chen CH and Yu ML Evolution of interferon-
based therapy for chronic hepatitis C Hepat Res
Treat 2010 20101409-53
5 Samuel B Bashar A Eric D et al US
Multicenter Pilot Study of Daily Consensus
Interferon (CIFN) Plus Ribavirin for ldquoDifficult-
to-Treatrdquo HCV Genotype 1 Patients Digestive
Diseases and Siences 201156880-8
6 Samuel S and Ayman A Predicting antiviral
treatment response in chronic hepatitis C how
accurate and how soon Journal of Antimicrobial
Chemotherapy 200151487-91
7 Rau M Baur K and Geier A Host Genetic
Variants in the Pathogenesis of Hepatitis C
Viruses 2012 4 3281ndash302
8 Tanaka Y Nishida N Sugiyama M et al
Genome-wide association of IL28B with response
to pegylated interferon-alpha and ribavirin
therapy for chronic hepatitis C Nat Genet
2009411105-9
9 Thomas DL Thio CL Martin MP et al Genetic
variation in IL28B and spontaneous clearance of
hepatitis C virus Nature 2009 461798-801
10 Ge D Fellay J Thompson AJ et al Genetic
variation in IL28B predicts hepatitis C treatment-
induced viral clearance Nature 2009 461399-
401
11 Suppiah V Moldovan M Ahlenstiel G et al
IL28B is associated with response to chronic
hepatitis C interferon-a and ribavirin therapy Nat
Genet 2009 411100-4
12 Kelly C Klenerman P Barnes E Interferon
lambdas the next cytokine storm Gut 2010
54102-9
13 Cavalcante LN Abe-Sandes K Angelo A et al
Il28b polymorphisms are markers of therapy
response and are influenced by genetic ancestry in
chronic Hepatitis C patients from an admixed
population Liver Int 2012 32 476ndash86
14 Dai CY Chuang WL Chang WY et al Tumor
Necrosis Factorndasha Promoter Polymorphism at
Position -308 Predicts Response to Combination
Therapy in Hepatitis C Virus Infection JID 2006
193 98ndash101
15 Cua IH Hui JM Bandara P et al Insulin
resistance and liver injury in hepatitis C is not
associated with virus-specific changes in
adipocytokines Hepatology 2007 46 66-73
16 Juran BD Atkinson EJ Larson JJ et al Carriage
of a tumor necrosis factor polymorphism
amplifies the cytotoxic T-lymphocyte antigen 4
attributed risk of primary biliary cirrhosis
evidence for a gene-gene interaction Hepatology
2010 52 223-9
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12
17 Nguyen-Khac E Houchi H Daoust M et al The
-308 TNF alpha gene polymorphism in severe
acute alcoholic hepatitis identification of a new
susceptibility marker Alcohol Clin Exp Res
2008 32 822-8
18 Wilson AG De Vries N Pociot F et al An allelic
polymorphism within the human tumor necrosis
factor a promoter region is strongly associated
with HLA A1 B8 and DR3 alleles J Exp Med
1993 177557ndash60
19 DrsquoAlfonso S and Richiardi PM A polymorphic
variation in a putative regulationbox of the TNFA
promoter region Immunogenetics 1994 39150ndash
4
20 Becker CM Fantini C Schramm H Lehr S Wirtz
A Nikolaev J et al TGF-suppresses tumor
progression in colon cancer by inhibition of IL-6
trans-signaling Immunity 2004 21 491-501
21 Galmozzi E Del Menico B Rametta R et al A
tetra-primer amplification refractory mutation
system polymerase chain reaction for the
evaluation of rs12979860 IL28B genotype J
Viral Hepat 2011 18628-30
22 Constantini PK Wawrzynowicz-Syczewska R
Clare M Interleukin-1 interleukin-10 and tumour
necrosis factor-alpha gene polymorphisms in
hepatitis C virus infection an investigation of the
relationships with spontaneous viral clearance and
response to alpha-interferon therapy Liver 2002
22 404-12
23 Bellanti F Vendemiale G Altomare E et al The
Impact of Interferon Lambda 3 Gene
Polymorphism on Natural Course and Treatment
of Hepatitis C Clin Dev Immunol
20122012849373-82
24 El-Awady M Mostafa L ATabll A et al
Association of IL28B SNP with Progression of
Egyptian HCV Genotype 4 Patients to End Stage
Liver Disease Hepat Mon 2012 12 271ndash7
25 Jung Y Kim J Ahn S et al Role of Interleukin
28B-related Gene Polymorphisms in Chronic
Hepatitis C and the Response to Antiviral
Therapy in Koreans J Clin Gastroenterol 2013
24 360-7
26 McCarthy J Li H Thompson A et al Replicated
association between an IL28B gene variant and a
sustained response to pegylated interferon and
ribavirin Gastroenterology 2010 38 2307-14
27 Mangia A Thompson AJ Santoro R et al
Limited use of interleukin 28B in the setting of
response-guided treatment with detailed on-
treatment virological monitoring
Hepatology 2011 54772-80
28 Mangia A Thompson AJ and Santoro R An
IL28B polymorphism determines treatment
response of hepatitis C virus genotype 2 or 3
patients who do not achieve a rapid virologic
response Gastroenterology 2010 139 821-7
29 Derbala M Rizk N Shebl F et al Interleukin-
28 and hepatitis C virus genotype-4 Treatment-
induced clearance and liver fibrosis World J
Gastroenterol 2012 187003-8
30 Pasha HF Radwan MI Hagrass HA et al
Cytokines genes polymorphisms in chronic
hepatitis C impact on susceptibility to infection
and response to therapy Cytokine 2013 61 478-
84
31 Yee LJ Tang J Gibson AW et al Interleukin 10
polymorphisms as predictors of sustained
response in antiviral therapy for chronic hepatitis
C infection Hepatology 2001 33708ndash12