Metodiche per lo studio della funzione piastrinica

Marco Cattaneo Medicina 2, ASST Santi Paolo e Carlo Dipartimento di Scienze della Salute

Università degli Studi di Milano Milano, Italy

Metodiche per lo studio della funzione piastrinica

• Diagnosi dei difetti congeniti della funzione piastrinica

• Monitoraggio della terapia antiaggregante (??)

Diagnosi dei difetti di funzione piastrinica

Test globali dell’emostasi primaria

Elevata variabilità Operatore-dipendente Impreciso Invasivo Poco sensibile Non utile nell’algoritmo diagnostico

Tempo di emorragia

Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo

Screening per VWD o Difetti di Funzione Piastrinica

Prolungato Normale

Tempo di emorragia

Paziente con sanguinamenti mucocutanei

Screening per VWD o Difetti di Funzione Piastrinica

Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo

Screening per VWD o Difetti di Funzione Piastrinica

Prolungato Normale

Tempo di emorragia

Paziente con sanguinamenti mucocutanei

Screening per VWD o Difetti di Funzione Piastrinica

Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo

Paziente con sanguinamenti mucocutanei

Screening per VWD o Difetti di Funzione Piastrinica

In Vivo PFA-100

capillary

aperture 150µm

epinephrine or

ADP

FLOW

sub-endothelium

platelet plug

endothelium

- 40 mbar

collagen

membrane

platelet plug

0

20

40

60

80

100

PCE PCA TE PCE PCA TE PCE PCA TE

PFD VWD

Difetti di coagulazione

75

33 38

86 86

29

14 3 14

14 14 0

Podda et al, JTH 2007

Percentuale di pazienti con valori patologici (tempi prolungati)

Screening per VWD

Prolungato

PFA-100® C-ADP

Paziente con sanguinamento mucocutaneo

Screening per PFD

VWD No VWD

Screening per PFD

Normale

Screening per VWD

PFD No PFD

Screening per VWD

Prolungato

PFA-100® C-ADP

Paziente con sanguinamento mucocutaneo

Screening per PFD

VWD No VWD

Screening per PFD

Normale

Screening per VWD

PFD No PFD

?? Da validare!!

Aggregometria a trasmissione di luce (LTA)

LTA – PRP citratato

Boneu & Cazenave

Recommendations • LTA is clinically useful for the study of subjects

with bleeding disorders • LTA should NOT be used for the identification of

subjects at risk for thrombosis • LTA should NOT be used to monitor subjects on

anti-platelet therapy

Recommendations: assessing the quality of PRP

Grossly hemolyzed samples should be discarded If the sample tested is lipemic, the final report should indicate

this It is necessary to check the platelet count of the PRP sample tested The results of LTA studies could be inaccurate when the platelet

count in the PRP samples is lower than 150 x 109/L, therefore, caution should be taken when interpreting abnormal results in samples with low platelet counts

PRP with low platelet counts may be tested to exclude severe platelet function disorders (BSS, type 2B and platelet type von Willebrand disease)

Platelet count of PRP samples should NOT be adjusted to a standardized value with autologous PPP (uncertain for PRP samples with platelet counts > 600 x 109/L)

Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate®)

LTA

p=NS p=NS p=NS

Femia et al, JTH 2013

Recommendations: Methodology - 1

LTA studies must include a known normal subject, run in parallel with the subject(s) under study

After centrifugation, PRP samples should be allowed to sit at room temperature for 15 min before testing

PRP should be used to set 0% light transmission in the aggregometer

Autologous PPP should be used to set 100% light transmission in the aggregometer

LTA studies should be performed at 37°C During LTA testing, PRP samples should be constantly stirred at

1,000 rpm using a disposable stirrer, unless otherwise specified by the manufacturer of the aggregometer

Before adding an agonist, baseline tracings for LTA should be observed for oscillations and stability for at least 1 minute

The volume of agonist added for LTA should be consistent, and never more than 10% of the total sample volume

Platelet aggregation should be monitored for: - a minimum of 3 minutes after adding an agonist - a minimum of 5 minutes after adding an agonist that does not

cause maximal aggregation by 3 minutes with most control samples - a minimum of 10 minutes after adding an agonist that does not

cause maximal aggregation by 5 minutes with most control samples LTA studies should be completed within a maximum of 4 hours

after blood sampling

Recommendations: Methodology - 2

Recommendations: agonists

The following platelet agonist should be used for diagnostic LTA studies: ADP: 2 µM (higher concentrations if abnormal results with 2 µΜ) Epinephrine: 5 µM (higher concentrations if abnormal results with 5

µΜ) Collagen: 2 µg/mL (Horm collagen) (higher concentrations if

abnormal results with 2 µg/mL) Thrombin Receptor Activating Peptide (TRAP): 10 µM (higher

concentrations if abnormal results with 10 µΜ) The thromboxane A2 mimetic U46619: 1 µΜ (higher concentrations

if abnormal results with 1 µΜ) Arachidonic acid: 1 mΜ (higher concentrations if abnormal results

with 1 mΜ) Ristocetin: 1.2 mg/mL

In case platelet agglutination induced by Ristocetin 1.2 mg/mL is normal, testing should be repeated using Ristocetin 0.5-0.7 mg/mL

In case platelet agglutination induced by Ristocetin 1.2 mg/mL is absent, testing should be repeated using Ristocetin 2 mg/mL.

Recommendations: evaluation and reporting of results

The platelet aggregation tracing should be evaluated based on: - presence of shape change - length of the lag phase - slope of aggregation - maximal amplitude or % aggregation - amplitude or % aggregation at the end of the observation - disaggregation - visual examination of the aggregation tracings The presence of a "secondary wave” induced by epinephrine should

be evaluated Studies completed more than 4 hours after blood collection should

be reported with a comment of this Clinical laboratories must establish an appropriate reference interval

and validate test performance with each lot of reagents

ADP Collagen AA Ristocetin

Normal

Glanzmann

Bernard -Soulier

SPD

Aspirin

Von Willebrand

temps trans

mis

sion

lum

ineu

se

LTA – PRP citratato

ADP Collagen AA Ristocetin

Normal

Glanzmann

Bernard -Soulier

SPD

Aspirin

Von Willebrand

temps trans

mis

sion

lum

ineu

se

LTA – PRP citratato

Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency:

studies in one hundred six patients

HK Nieuwenhuis, JW Akkerman, JJ Sixma

Blood 1987; 70:620-623

Normal Normal + ASA II-4 II-6 PSD

20 2 4

4 20

2

2 4

20

20

4 2

2

4

20

2

4

20

20

Platelet aggregation (upper tracings) and secretion (lower tracings) induced by ADP at the indicated concentrations (μM),

obtained with the lumiaggregometer

δ-SPD

Algoritmo diagnostico dei difetti di secrezione piastrinica

Secrezione piastrinica

Anormale Normale

STOP Dosaggio del contenuto dei granuli

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

δ-Granule MAO

5HIAA

Normal platelet

VMAT

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Tests to measure platelet serotonin (5HT) secretion

1. Secretion of 14C-5HT (or 3H-5HT) from pre-loaded platelets (considered the gold standard)

2. Tests to measure secreted endogenous 5HT: – Fluorimetric assay using ortho-phtalaldehyde – ELISA – HPLC, coupled to electrochemical or

fluorescence detection – Liquid chromatography tandem-mass

spectrometry (LC-MS)

Tests to measure platelet serotonin (5HT) secretion – General principles

1. Both total platelet content of 5HT and secreted 5HT should be measured (total and secreted radioactivity in case of 14C-5HT or 3H-5HT)

2. Values expressed as percent secretion of total content

3. Platelet stimulation should be performed in the presence of a SERT inhibitor (usually, imipramine), in order to prevent the reuptake of secreted 5HT

Tests to measure platelet serotonin (5HT) secretion – advantages and disadvantages

Method Advantages Disadvantages

Radiolabeled 5HT - gold standard - simple

- use of radioisotopes - not suitable for patients with delta granules deficiency

o-phthaldehyde - low cost - time consuming

ELISA - expensive

HPLC - accurate, precise - expensive instrumentation - experienced personnel

LC-MS - accurate, precise - expensive instrumentation - experienced personnel

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

δ-Granule MAO

5HIAA

Piastrina normale

VMAT

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

VMAT δ-Granule

MAO 5HIAA

MAO 5HIAA

MAO 5HIAA

Piastrina δ–SPD

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

δ-Granule MAO

5HIAA

Piastrina normale

* * *

*

*

* * *

* VMAT *

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

δ-Granule

* * *

*

*

* MAO 5HIAA *

*

* VMAT

MAO 5HIAA

MAO 5HIAA *

*

Piastrina δ–SPD

*

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

δ-Granule MAO

5HIAA

Piastrina normale stimolata

* * *

*

*

* * *

* VMAT

*

* *

*

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Trasporto e localizzazione della serotonina (5HT) nelle piastrine

5HT

SERT

δ-Granule

* * *

*

*

* MAO 5HIAA *

*

* VMAT

MAO 5HIAA

MAO 5HIAA *

*

Piastrina δ–SPD stimolata

*

*

SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid

Aggregazione piastrinica

Metodo impedenziometrico

firm adhesion and aggregation of platelets on the sensor surface enhances the electrical resistance between the 2 sensor wires

Principle of Multiplate® analysis

aggr

egat

ion

[AU

]

time [min]

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5

test 1 test 2

MULTIPLATE

Normale

Clopidogrel

aggr

egat

ion

[AU

]

time [min]

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5

test 1 test 2

Ligh

t tra

nsm

issi

on (%

)

0 0 1 2 3 4 5

time [min]

MULTIPLATE

Normale

Clopidogrel

LTA

Normale

Clopidogrel

aggr

egat

ion

[AU

]

time [min]

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5

test 1 test 2

Ligh

t tra

nsm

issi

on (%

)

0 0 1 2 3 4 5

time [min]

MULTIPLATE

Normale

Clopidogrel

LTA

Normale

Clopidogrel

aggr

egat

ion

[AU

]

time [min]

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5

test 1 test 2

Ligh

t tra

nsm

issi

on (%

)

0 0 1 2 3 4 5

time [min]

MULTIPLATE

Normale

Clopidogrel

LTA

Normale

Clopidogrel

aggr

egat

ion

[AU

]

time [min]

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5

test 1 test 2

Ligh

t tra

nsm

issi

on (%

)

0 0 1 2 3 4 5

time [min]

MULTIPLATE

Normale

Clopidogrel

LTA

Normale

Clopidogrel

Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate®)

LTA

p=NS p=NS p=NS

Femia et al, JTH 2013

Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate®)

LTA

Multiplate®

p=NS p=NS p=NS

p<0.001 p<0.001 p<0.001

Femia et al, JTH 2013

Correlazione tra aggregazione piastrinica in sangue intero (Multiplate) e ematocrito

Kakouros et al, JTH 2014

Diagnostic performance for PFD in 109 children with bleeding history: LTA vs Multiplate

Test N. of patients with abnormal results

LTA 15 Multiplate 3

Haas et al, Platelets 2018

Response variability to Clopidogrel

The solution?

“Laboratory monitoring of antiplatelet treatment”: increase the dose of Clopidogrel (use another drug)

in patients with HTPR (based on the results of platelet function tests)

A MEANS to be used if of proven efficacy and safety!

1. Identification of the most accurate laboratory test

2. Standardization of pre-analytical and analytical variables [??]

3. Identification of universal cut-off values [??]

4. Clinical validation [??]

Validation of laboratory monitoring of clopidogrel treatment

Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA):

in vivo vs in vitro experiments

Danese et al, JTH in pres

in vivo

in vivo

in vitro

in vitro

-1.5 -1.0 -0.5 0.00

20

40

60

80R2: 0.087

Multiple Electrode Aggregometry (ADP)

CAM (µmol/L)

agg

rega

tion

units

(U)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80

100R2:0.580

Multiple Electrode Aggregometry (ADP)

CAM (µmol/L)

aggr

egat

ion

units

(U)

-1.5 -1.0 -0.5 0.00

20

40

60

80R2: 0.175

Multiple Electrode Aggregometry (ADP+PGE 1)

CAM (µmol/L)

aggr

egat

ion

units

(U)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80R2: 0.548

Multiple Electrode Aggregometry (ADP+PGE 1)

CAM (µmol/L)

agg

rega

tion

units

(U)

A B

C D

0.05 0.1 0.5 1.0

0.05 0.1 0.5 1.0 5.0 10.0 15.0 0.05 0.1 0.5 1.0

0.05 0.1 0.5 1.0 5.0 10.0 15.0

Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA):

in vivo vs in vitro experiments

Danese et al, JTH in pres

in vivo

in vivo

in vitro

in vitro

-1.5 -1.0 -0.5 0.00

20

40

60

80R2: 0.087

Multiple Electrode Aggregometry (ADP)

CAM (µmol/L)

agg

rega

tion

units

(U)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80

100R2:0.580

Multiple Electrode Aggregometry (ADP)

CAM (µmol/L)

aggr

egat

ion

units

(U)

-1.5 -1.0 -0.5 0.00

20

40

60

80R2: 0.175

Multiple Electrode Aggregometry (ADP+PGE 1)

CAM (µmol/L)

aggr

egat

ion

units

(U)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80R2: 0.548

Multiple Electrode Aggregometry (ADP+PGE 1)

CAM (µmol/L)

agg

rega

tion

units

(U)

A B

C D

0.05 0.1 0.5 1.0

0.05 0.1 0.5 1.0 5.0 10.0 15.0 0.05 0.1 0.5 1.0

0.05 0.1 0.5 1.0 5.0 10.0 15.0

-1.5 -1.0 -0.5 0.00

20

40

60

80R2: 0.087

Multiple Electrode Aggregometry (ADP)

CAM (µmol/L)

agg

rega

tion

units

(U)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80

100R2:0.580

Multiple Electrode Aggregometry (ADP)

CAM (µmol/L)

aggr

egat

ion

units

(U)

-1.5 -1.0 -0.5 0.00

20

40

60

80R2: 0.175

Multiple Electrode Aggregometry (ADP+PGE 1)

CAM (µmol/L)

aggr

egat

ion

units

(U)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50

20

40

60

80R2: 0.548

Multiple Electrode Aggregometry (ADP+PGE 1)

CAM (µmol/L)

agg

rega

tion

units

(U)

A B

C D

0.05 0.1 0.5 1.0

0.05 0.1 0.5 1.0 5.0 10.0 15.0 0.05 0.1 0.5 1.0

0.05 0.1 0.5 1.0 5.0 10.0 15.0

Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA):

in vivo vs in vitro experiments

Danese et al, JTH in pres

in vivo

in vivo

in vitro

in vitro

Vasodilator Stimulated Phosphoprotein (VASP) Assay for the Measurement of P2Y12 Antagonism

In the presence of both PGE1 and ADP, VASP-P is directly proportional to the degree of P2Y12 antagonism

Modified from Cattaneo in PLATELETS (Michelson, 2nd ed, 2007)

ADP Antagonist

-1.5 -1.0 -0.5 0.0 0.5 1.00

25

50

75

100

in vivo study R2: 0.871in vitro study R2: 0.847

CAM (µmol/L)

PRI (

%)

0.05 0.1 0.5 1.0 5.0 10.0

Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with PRI (VASP phosphorylation assay):

in vivo vs in vitro experiments

Danese et al, JTH in pres

1. Identification of the most accurate laboratory test [??]

2. Standardization of pre-analytical and analytical variables [??]

3. Identification of universal cut-off values [??]

4. Clinical validation [??]

Validation of laboratory monitoring of clopidogrel treatment

1. Identification of the most accurate laboratory test [??]

2. Standardization of pre-analytical and analytical variables [??]

3. Identification of universal cut-off values [??]

4. Clinical validation

Validation of laboratory monitoring of clopidogrel treatment