SocietàItaliana diCitometria

PROCEEDINGS OF THE XXXI NATIONAL CONFERENCEOF CYTOMETRY

Palazzo DucaleLucca, Italy

October 8-11, 2013

EDITED BYR. DE VITA and G. MAZZINI

Scientific BoardA. Biondi (Monza)P. Bonara (Milano)A. Bosi (Firenze)A. Calugi (Roma)E. Cannizzo (Lucca)R. Chianese (Ivrea)M. Danova (Vigevano)L. Del Vecchio (Napoli)M.G. Della Porta (Pavia)A. Fattorossi (Roma)D. Fenoglio (Genova)A. Kunkl (Genova)F. Lanza (Cremona)E. Marcenaro (Genova)L. Petrini (Pisa)M. Rocchi (Bari)A. Russo (Palermo)M. Spanò (Roma)C. Usai (Genova)

Scientific CoordinatorsR. Casotti (Napoli)I. D’Agnano (Roma)S. Lucretti (Roma)L. Zamai (Urbino)

Organizing CommitteeG. Gaipa (Monza)R. De Vita (Roma)E. Erba (Milano)G. Mazzini (Pavia)G. Pirozzi (Napoli)

SOCIETÀ ITALIANA DI CITOMETRIAc/o Unità Biologia delle Radiazioni e Salute dell'Uomo

ENEA Centro Ricerche Casaccia s.p. 016Via Anguillarese, 301 - 00123 Roma - tel. 06 30484671 - fax 06 30484891

e-mail: gic@enea.it - http://biotec.casaccia.enea.it/GIC/

SUPPORTED BY ENEA - AGENZIA NAZIONALE PER LE NUOVE TECNOLOGIE,L’ENERGIA E LO SVILUPPO ECONOMICO SOSTENIBILE

XXXI National Conference of the Italian Society of Cytometry GICOctober 8-11, 2013

Lucca, Italy

The XXXI Italian Society of Cytometry (GIC) bi-annual meeting had been held in Lucca (Tuscany,Italy) from October 8th to 11th. 2013.All abstracts were carefully reviewed by the Scientific Program Committee and published here in fulland categorized by scientific tracks such as cell cycle and apoptosis; environmental sciences andtoxicology, hematology, immunology, methodology-technology, and oncology.To date there are over 600 members actively involved in educational programs, promotion of quali-ty controls programs, drafting/validation of guidelines and accreditation, providing information forpeople involved that actively work in the field of basic and applied cytometry.

This years the Conference had been preceded by a half a day satellite event devoted to the establi-shment of the “Professional Certification GIC Register” which will be committed to test, train, cer-tify and monitor the professional levels of the “Italian Cytometrists” under the responsibility of GIC.At present more than 50 members have applied to be enrolled in the formative training structure.After the series of “dedicated seminars” the attendees had the opportunity to discuss with the “EntryCommittee” the consistency of the basic requirements they have declared in a preliminary “entryform”. Among all the CV items applicants should have a minimum of three years practical expertisein the field. Anyone passing this entry level will be examined by a formal “Examination Committee”that formally assignee the title of “Qualified Cytometrist”. This new GIC initiative had been verymuch appreciated by the members.Regarding the scientific program this year the Scientific Committee selected 31 abstracts for oral pre-sentation among those submitted (110). Plenary sessions included 9 invited lectures focused on thecontribute of cytometry in different application fields, highlighting the interdisciplinary profile ofGIC. The opening lecture was given by Prof. Dario Campana (University of Singapore) and wasfocused on how flow cytometry can guide treatment decisions in acute leukemia. This outstandingtalk offered a rational and fascinating overview on the role of flow cytometry in the diagnosis andclinical management of acute leukemias including the future perspectives of monitoring the patent’sindividual response to therapy in modern clinical trials.Each Parallel session, offered along three days, was tailored on the traditional topics such as immu-nology, oncology, hematology, methodology, and environmental sciences with cytometry as the com-mon denominator. Several invited and selected lectures were given in order to bring the discussionon the more emerging and promising aspects of flow cytometry.

Again in the “core” of the Society activities a special session had been programmed to the reports ofthe ongoing GIC Projects:a) “Professional Qualification”: R. De Vita summarized the strategic importance in Italy (as eve-rywhere in the world) to apply a “formal-serious-system” to take care and guarantee the highProfessional level of people (Beginners as well as Seniors) involved in the area of analyticalcytometry especially those having clinical impacts. The “national-governance” should have inmind the patients-care and the scientific society may have a crucial role in these initiatives.

b) “The role of Flow Cytometry in the immunophenotyping of Acute Leukemia.” G. Gaipa madean exhaustive details of what the board of this Project did in the recent past and what the enrol-

led “Expert Committee” is doing in the evaluation of the selected literature in order to be ableto define in the future step of the activity witch is the real impact of Flow Cytometry in thisvery important and promising area of application of this technology.

c) “Standardization of a Flow Cytometry approach for the evaluation of Endothelial Circulatingcells and their progenitors”. M. Della Porta reported the activities of the group of labs (spre-ad around the north Italy) practically involved in the analyses of reference samples prepared inPavia and rapidly sent to the participants. The very ambitious Original Design involving a verylarge number of markers did results in interlaboratories CVs of the preliminary samples extre-mely wide. In a successive step and thanks to the “analytical-instrumental-setting” pre-definedby the Committee the actual output of the trial is going to be greatly improved. The support ofthe companies (Beckman Coulter and Miltenyi Biotec) had been extensively acknowledgedwhile the cost of the Project was out of the financial possibility of the GIC Society.

d) “Quality assessment of the cytometric Laboratory”. R. Chianese made a summary of the acti-vity she has devoted first to introduction and spread around the members of the Society theconcept of the “Quality” in the cytometry labs and therefore to the way to improve and main-tain the minimum level. The preliminary phase of this initiative have enrolled only few labs butthanks to the capillary sensibilization action (carried out with the support of the GIC secretaryoffice) the situation had been significantly improved and there is now a large group of users allaround Italy interested to be trained in quality up-grade. Very important will be the forecastinteractions between this Project and the one dealing with “Professional Qualification” whilethe “Quality items” will be certainly one of the mile stones to be upgraded in the professionalculture of the next new generation of cytometrists.

The Conference had also been characterized by two special symposium (topics were “Mimimal resi-dual disease” and “Circulating tumor cells”), in which selected experts from the main ItalianSocieties in the fields of Hematology and Oncology has brought their top contribution.Poster session included 79 contribution and a special committee have selected 2 Poster award win-ner in each category.A substantial contribution has been provided by the principal companies in the field which had beenlocated in a large exhibition area inside the conference center.The President conclusive remarks included the great satisfaction for the very good scientific level ofall the sessions with particular reference to the “Posters” definitely established as the most commonway to introduce novel data and discoveries in science.

President Past PresidentG. Gaipa F. Lanza

EDITED BYR. DE VITA and G. MAZZINI

table of contents

Invited Speakers 5

Session ICell Cycle and Apoptosis 11

Session IIEnvironmental Sciences and Toxicology 15

Session IIIHematology 20

Session IVImmunology 31

Session V 39

Session VIOncology 45

Authors’ index 52

FLOW CYTOMETRY TO GUIDE TREATMENT DECISIONSIN ACUTE LEUKEMIACampana D.Department of Paediatrics, Yong Loo Lin School ofMedicine, National University of Singaporepaedc@nus.edu.sg

In addition to its established application to the diagnosisof hematologic malignancies, flow cytometry can con-tribute in many meaningful ways to patient clinical man-agement. For example, leukemia subtypes that are asso-ciated with a poor response to therapy can be identifiedbased on their cell marker profile. Early T-cell precursoracute lymphoblastic leukemia (ETP-ALL) is one suchtype, characterized by a defined immunophenotype.Another area where flow cytometry can help providingvaluable prognostic information is in the detection of min-imal residual disease (MRD), i.e., leukemic cells unde-tectable by standard morphological methods. The mostreliable approaches to study MRD in acute leukemia areflow cytometric detection of aberrant immunophenotypesand polymerase chain reaction amplification of rear-ranged immunoglobulin and T-cell receptor genes andchromosomal breakpoints. These methods can detect 1leukemic cell among 10,000 or more normal bone mar-row or peripheral blood cells. The results obtained withthe two methods in childhood ALL are highly concordant.In patients with acute myeloid leukemia (AML), flowcytometry is the only method that allows MRD monitoringin the majority of patients. Prospective studies of MRD inpatients with newly diagnosed ALL and AML have shownthat presence of MRD detected by flow cytometry in bonemarrow is strongly and independently associated with ahigher risk of relapse. There is strong evidence that MRDlevels before hematopoietic stem cell transplantation inpatients with ALL are closely related to the risk of relapsepost-transplant; emerging data indicates that this correla-tion also occurs in AML. The discovery of new markers todistinguish leukemic from normal cells should improvethe reliability and sensitivity of MRD studies by flowcytometry, and further widen its application.

PROBING THE BRAIN BY LIGHT: MEASURING INTRA-CELLULAR PH AND CHLORIDE CONCENTRATION BYMEANS OF IN VIVO TWO-PHOTON SPECTROSCOPY1,2Ratto GM., 2,1Artoni P., 1Brondi M., 3Idilli A., 2,1Landi S.,2Luin S., 2Negro A., 3Paredes J.M. 2,4Sulis Sato S.,4Cancedda L., 3Arosio D.1NEST, Istituto Nanoscienze CNR, NEST, 2ScuolaNormale Superiore, 3Istituto Biofisica CNR, Trento 4IIT,Genova, ItalyGianmichele.ratto@sns.it

The correct operation of the brain is assured by the finetuning of excitation and inhibition between neurons. Acrucial determinant of the strength of inhibition is theintracellular concentration of Chloride [Cl]i, with low levelsof [Cl]i in the postsynaptic neuron indicating a strong inhi-bition. It is becoming clear that disruption in Cl homeosta-sis is implicated in a great varieties of brain dysfunctions,but, up to now, no suitable tool has been available tomeasure [Cl]i in the intact brain. ClopHensor is a ratio-metric non-FRET biosensor based on a highly Cl- and

pH-sensitive GFP variant (E2GFP), fused with a red fluo-rescent protein, which fluorescence is independent on pHand [Cl-]. In this study our aim is to use this sensor in theanesthetized animal by means of 2-photon in vivo spec-troscopy. In order to do this, we have to: a) understandthe 2-photon excitation properties of the indicator and b)determine an imaging protocol that allows the independ-ent ratiometric estimates of pH and Cl-. Two-photonspectroscopy of the recombinant sensor and of culturedcells is used to build a calibration model that allow tocompute pH and [Cl]i from the observed spectra.However, the transport of these calibration data to the invivo imaging is not straightforward: the brain is not trans-parent and both absorption and scattering of light arestrongly wavelength-dependent. Therefore the spectralsignature of the sensor is strongly distorted by the tissueoverlying the measured cells. Here, we will present a pro-cedure that allow to compute the spectral distortion of thesensor caused by the brain tissue. In this way we canestimate absolute [Cli and pH in vivo in the intact brain innormal and pathological conditions. By using two modelocked laser, it is possible to perform dynamic measuresof Cl fluxes during brain operation, thus opening a newwindow on our understanding of neuronal inhibition.

CHROMOSOME GENOMICS: A QUANTUM JUMP INANALYZING COMPLEX PLANT GENOMESDolezel J,1 Lucretti S.,21Institute of Experimental Botany, Centre of the RegionHaná for Biotechnological and Agricultural Research,Olomouc-Holice, Czech Republic – dolezel@ueb.cas.cz2ENEA - Italian National Agency for New Technologies,Energy and Sustainable Economic Development,Casaccia Research Center, Rome, Italysergio.lucretti@enea.it

Plants exhibit large variation in genome size and a num-ber of them, including wheat have genomes many timeslarger than that of human and comprising many billionsbase pairs. Genomes expanded mainly due to repetitiveDNA and polyploidy. DNA sequence redundancy ham-pers gene mapping and cloning as well as genomesequence assembly. An attractive approach to overcomethis difficulty is to dissect genomes to small parts andanalyze them separately. Chromosomes are natural unitsof nuclear genomes and mitotic metaphase chromo-somes can be sorted by flow cytometry in a number ofplants. The sorted chromosomes have been used to sim-plify physical genome mapping using PCR and FISH,develop DNA markers from particular chromosomes andconstruct BAC libraries. The availability of chromosomeBAC libraries facilitates construction of ready to se-quence physical maps of complex genomes, and theirproduction has underpinned the international effort tosequence the wheat genome. The spread of next gener-ation sequencing methods opened new horizons for chro-mosome genomics. Chromosome survey sequencing is apowerful way to study molecular composition of individualchromosomes, identify majority of genes, develop virtuallinear gene order models and improve whole genomesequence assemblies. Chromosome sorting was initiallylimited by inability to discriminate chromosomes with thesame DNA amount. One solution is to use cytogeneticstocks in which chromosome size has been changed dueto translocations and/or deletions. Moreover, a carefulselection of sort windows enables to obtain fractionshighly enriched for chromosomes of interest. A morerefined approach relies on labeling specific chromo-somes prior to flow cytometry using fluorescence in situ

Invited Speakers

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ABSTRACTS FROM THE XXXI CONFERENZA NAZIONALE DI CITOMETRIA

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ABSTRACTS FROM THE XXXI CONFERENZA NAZIONALE DI CITOMETRIAhybridization in suspension (FISHIS). Thus, chromo-somes can be sorted from various materials and chromo-some genomics has been increasingly used to facilitatethe analysis of large and polyploid genomes where wholegenome approaches may fail.

SILICON OPTICAL MICROCHIP FOR NEW CYTOMETRICAPPROACHES IN CELLBIOLOGYMazzini G.IGM-CNR e Dipartimento di Biol. L. Spallanzani, UniPV,Pavia Italymazzi@igm.cnr.it

Investigations of cell cultures in 3D microenvironmentshave attracted an increasing interest by research scien-tists in the biomedical area. Although cells typically residein vivo in a three-dimensional (3-D) environment, most ofwhat is known about cells has been derived from culturesperformed on flat surfaces, such as plastic Petri dishes orglass slides. Therefore, there is an increasing interest ininvestigating cell cultures on 3-D matrices, also known asscaffolds, since these structures can have major effectson cell behavior with regard to adhesion proliferation, dif-ferentiation and, also, apoptosis.We have recently proposed the use of silicon devicesbased on a well-ordered material as a three-dimensionalsupporting matrix for biological nanostructures and foroptofluidic applications. A cell-selective silicon microincu-bator, that incorporates a vertical, high aspect-ratio (HAR)silicon photonic crystal (PhC) as core element, has beensuccessfully demonstrated for performing cell cultures in a3-D microenvironment[1] [2]. HAR PhCs consist of period-ic arrays of parallel ≈ 3 µm-thick silicon walls separated by≈ 5 µm-wide, 50 µm-deep air gaps, fabricated by electro-chemical micromachining (ECM) of <100>-oriented siliconwafers. PhCs are artificial materials characterized by thepresence of photonic bandgaps, i.e., wavelength intervalsin which the propagation of the electromagnetic field insidethe material, in our case in direction orthogonal to the sili-con walls, is prohibited. The definition of interaction effectsbetween PhCs and cell cultures represents a first signifi-cant step toward the fabrication of a new-concept cell-based optical biosensor, in which cells grow and proliferateinto HAR PhC transducers, for direct label-free opticalmonitoring of cellular activities.As a first step we have investigated the peculiar behav-iour of different cell lines belonging to two differentembryonic origin such as the epithelial versus the mes-enchimal. The experimental results strongly support ourstatement that only cells with mesenchymal phenotypeposses the peculiar ability to actively colonize the gaps,adherent to the inner vertical surfaces of the silicon walls,thus demonstrating that these HAR micromachined struc-tures, as microincubators, exhibit a sort of cell selectivity.These findings support our idea to use silicon PhC asboth cell-selective microincubator and optical “labell-free”transducer of cell morphology for advanced biomedicalapplications [3]. Variation in cell morphology as well asadhesion/detachement to silicon walls are easily probedby the system acting as a core point of a family of lab-on-chip respectively dedicated to the EMT (probing morphol-ogy) and to apoptosis (probing cell detachement).Applications in the fields of tumor biology as well as in thedrug-discovery will be described.Very promising are the implication of cell biomechanicsand transforming potential (cancer mechanobiology) beingour silicon PhC a suitable device to study the different abil-ity of cancer cell to penetrate the narrow microcavityaccording to their biological aggressivity. Preliminary

experiments on tumour cell lines derived from a variety oftumors of various grade of malignancy have confirmed thatthose deriving from high grade are to enter the PhC micro-cavity much more than those from low grade.Final step of our future researchers is again in the area ofclinical oncology: the search and evaluation of the“Circulating Tumor Cells” in the peripheral blood of tumorpatients seem to be very promising to be performed withour technology. CTC are in fact characterized by peculiar“bio-mechanical properties” to have the ability to strechinside the microcapillary, where they undergo the EMTprocess and finally they posses (by definition) the highbiological aggressivity to be able to originate in a distantsite the metastasis of the original tumor. Work partiallysupported by Fondazione Cariplo, grant no. 2011-0308.

PERIPHERAL BLOOD DENDRITIC CELLS IN HUMANHEALTHY AND PATHOLOGICAL PREGNANCYDella Bella S.,1,2 Calcaterra F.1,2

1University of Milan, Department of Medical Biotechnologiesand Translational Medicine, Milan, Italy; 2Humanitas Clinicaland Research Center, Lab of Clinical and ExperimentalImmunology, Rozzano (MI), Italysilvia.dellabella@unimi.it

Pregnancy provides a major challenge to the maternalimmune system, which must tolerate fetal alloantigensencoded by paternal genes for allowing the fetus to growand develop in the uterus despite being recognized.Dendritic cells (DCs) are widely distributed potent anti-gen-presenting cells that bridge the arms of innate andadaptive immunity. They can either promote or preventimmune activation, thus playing a central role in the con-trol of immune tolerance. DCs in pregnant women havebeen characterized mainly at the maternal-decidual inter-face, where their state of activation has emerged as oneof the key players influencing the feto-maternal immuno-logical equilibrium. Growing evidences indicate that dur-ing pregnancy also DCs circulating in the peripheralblood, which is the most accessible source of DCs,undergo changes that may be relevant to the adaptationof maternal immune responses needed to allow fetalgrowth. Moreover, alterations of circulating DCs that maylikely reflect alterations occurring in the decidua havealso been described in pathological pregnancies, such asin pregnancies complicated by intrauterine growth restric-tion or pre-eclampsia.

RECEPTOR-MEDIATED REGULATION OF HUMAN NKCELL HOMING RECEPTOR-MEDIATED REGULATIONOF HUMAN NK CELL HOMINGMarcenaro E., Pesce S., Moretta A.Laboratorio di Immunologia Molecolare - Dipartimento diMedicina Sperimentale (DI.ME.S.) and Centro diEccellenza per le Ricerche Biomediche (CEBR),Università degli Studi di Genova, ItalyEmanuela.Marcenaro@unige.it

After recruitment in peripheral tissues by chemokine gra-dients, NK cells must undergo a priming process in orderto acquire full functional competence before migratingtowards lymph nodes. Priming takes place when NK cellsinteract with other innate immunity cell types, either resi-dent or recruited during inflammation (which release a setof relevant cytokines) as well as by the recognition ofvirus-infected or tumor target cells.In this context, the interaction of NK cells with Dendriticcell (DC) during the early phases after pathogen invasionappears to play a crucial role in the generation of down-

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ABSTRACTS FROM THE XXXI CONFERENZA NAZIONALE DI CITOMETRIAstream adaptive T cell immunity and in the acquisition ofparticular chemokine receptor involved in the homingprocess, such as CCR7. We showed that freshly isolatedperipheral blood KIR+ NK cells, as well as KIR+ NK cellclones, de novo express CCR7 upon co-culture withmature DC. As a consequence, they become capable ofmigrating in response to the CCR7-specific chemokinesCCL19 and/or CCL21. This mechanism is tightly regulat-ed by KIR-mediated recognition of HLA class-I. Analysisof NK cell clones revealed that KIR-mismatched but notKIR-matched NK cells acquire CCR7. Moreover, weshowed that KIR2DS1+ NK cells upon interaction withHLA-C2+ target cells acquire CCR7 and migrating prop-erties. These data have important implications in hap-loidentical haematopoietic stem cell transplantation(HSCT), in which KIR-mismatched NK cells may acquirethe ability to migrate to lymph nodes, where they can killrecipient’s antigen presenting cells (APCs) and T cellsthus preventing graft vs host (and host vs graft) reactions.

COMPARATIVE ANALYSIS OF FLOW CYTOMETRYAND HYSTOLOGY FOR THE EVALUATION OFLYMPHONODE BIOPSIESLanza F., Spedini P., Ongari M., Lazzari C., Pasini A.L.,Pinotti P., Tajana M., Fiamenghi C., Gusolfino D., Betri E.Section of Hematology and BMT Unit, “HistopathologyService, Cremona Hospital, Cremona, Italyf.lanza@ospedale.cremona.it

Flow cytometry (FC), is a qualitative and quantitativeanalysis technology; it characterizes cell populations at asingle cell level. FC is considered a powerful tool for thediagnosis of mature B-cell lymphoid neoplasms and mayassist in the diagnosis and classification of mature T- andNK-cell lymphoid tumors. Although FC is able to provideobjective and quantitative results, even on very smallsamples, it is not routinely applied in the evaluation ofsolid tissues. In this study, we evaluated the contributionof flow cytometric immunophenotyping along with cellblock immunocytochemistry as adjunct technique in thediagnosis and classification of lymphoproliferative disor-ders (mainly Non-Hodgkin and Hodgkin Lymphoma, asper the current WHO classification). The main aim of ourstudy was to classify the lymphomas as accurately aspossible based on FC assessment by immunophenotyp-ing. Our data showed that FL on tissue samples is a pow-erful adjunct to morphology and immunohistochemistry inthe diagnosis of several haematological malignanciesand may provide rapid and accurate diagnosis of lym-phomas.

FLOW CYTOMETRY IN THE CANCER RESEARCHLABORATORYD’Agnano I.Institute of Cell Biology and Neurobiology-CNR, Rome,Italy -igea.dagnano@cnr.it

In a post-genomic era, in which epigenetics is one of themajor player and the gene is put in its living context,rather than detached from its material matrix, the study ofthe cell in its whole becomes even more actual. In partic-ular, in the field of cancer research this approach appearsto be more appropriate for the understanding of such acomplex genetic diseases, thus allowing to get insightinto some obscure aspects of cancer pathogenesis suchas cancer cell dormancy, chemo-resistance, local anddistant relapses. Tumors are known to be characterizedby lack of cellular homogeneity originating from a numberof sources, including differential nutrient status due to

the de novo microcirculations of tumors, to infiltration ofnormal cells into the tumor, and to the hierarchicalnatures of the cell populations from which cancers arise,the so called tumor initiating cells (TIC). TIC are oftenderived from tissue stem cells and these cancer stemcells are characterized by resistance to most cytotoxictreatments and by a high metastatic rate.In this scenario flow cytometry is recognized as an invalu-able technology to study the complexity of cancer dis-ease. In recent years, major advances in single-cellmeasurement systems have included the introduction ofhigh-throughput versions of traditional flow cytometry thatare now capable of measuring intracellular network activ-ity and, thanks to the emergence of a variety of isotopelabels, can enable the tracking of a great variety of cellmarkers and components. Owing to its multiparametricnature flow cytometry can provide highly detailed infor-mation on any single cell in a heterogeneous population,thus facilitating our capacity to catalog and bring order tothe inherent diversity present in cancer cell populations.In addition, the wide applications provided by flow cytom-etry have the potential to reveal new insights into cancerbiology at the intersections of stem cell function, TIC andmultilineage tumor development. Its versatility and flexi-bility means it can be conducted in the preclinical andclinical setting, remaining at the forefront of both routineand exploratory cancer studies.

CD39 AND IMMUNOREGULATION: NOT ONLYREGULATORY CD4+ T CELLSFenoglio D.,1,3 Parodi A.,1 Battaglia F.,1 Kalli F.,1

Conteduca G.,1 Negrini S.,1 Serpero L.,4 Bernuzzi F.,5Borgonovo G.,2 Simonato A.,1,2 Traverso P.,1,2

Invernizzi P.,5 Uccelli A.,4 Filaci G.1,3

1Centre of Excellence for Biomedical Research; 2Dep ofSurgical Sciences; 3Dep of Internal Medicine; 4Dep ofNeuroscience, Rehabilitation, Ophthalmology, Genetics,Maternal and Child Health- University of Genoa, Italy;5Center for Autoimmune Liver Diseases, IRCCS IstitutoClinico Humanitas, Rozzano, Italydaniela.fenoglio@unige.it

CD39 is an ectoenzyme which mediates immunosuppres-sive functions catalyzing ATP degradation. It is not knownwhether CD39 is implicated in the activity of CD8+ regula-tory T lymphocytes (Treg). To this issue, we analyzedCD39 expression and regulatory function in both CD8+and CD4+CD25hi Treg in two diametrically opposed clinicalsettings, as autoimmunity and tumor immune escape. Inparticular, the study evaluated peripheral Treg cells infibrotic autoimmune disease and multiple sclerosis (MS);whereas in tumor context, CD39 expression and functionwas analyzed in peripheral blood as well as in tumor spec-imens from cancer patients. The results showed defects inthe CD8 + Treg subset combined or not with abnormalitiesregarding the CD4 + CD25 + Treg cells in autoimmune dis-eases. Moreover, although CD39 was found expressed byboth CD8+ (from the majority of healthy donors and tumorpatients) and CD4+CD25hi Treg, CD39 expression corre-lated only with suppression activity mediated by CD8+Treg. Importantly, CD39 counteraction remarkably inhibit-ed the suppression activity of CD8+ Treg (both fromperipheral blood and tumor microenvironment) indicatingthat CD39-mediated inhibition constitutes a prevalent hall-mark of their function. Collectively, these findings, unveilinga new mechanism of action for CD8+ Treg, provide newknowledge and new therapy target on both immune-medi-ated diseases and intratumoral molecular pathways relat-ed to tumor, immune escape.

ABSTRACTS FROM THE XXXI CONFERENZA NAZIONALE DI CITOMETRIA

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CELL COMMUNICATION AND APOPTOSIS INLYMPHOCYTE CELLSLuchetti F2, Canonico B.1, Arcangeletti M.2, Galli L1,Cesarini E.1, Zamai L.1, Papa S.2.1Department of Earth, Life and Environment, University ofUrbino “Carlo Bo”. 2Department of BiomolecularSciences, University of Urbino “Carlo Bo”, Urbino. Italyfrancesca.luchetti@uniurb.it

Communication with the environment is the basic princi-ple of any biological system, with the increasing complex-ity of higher organisms cell had to evolve several diversemechanisms to exchange informations crucial for tissueorganization and maintenance of the organism as awhole. To date, different mechanisms for the exchange ofmolecular information between cells have documented:the exchange of membrane fragments (trogocytosis), for-mation of tunneling nanotubes (TNTs) and release ofmicrovesicles (MVs). In our study, paring up flow cyto-metric and confocal microscopy approaches, we haveinvestigated apoptosis linked to the intercellular commu-nication pathway, in an early time-frame of stimulation.Jurkat cells and CD4+ T lymphocytes have been stainedwith different dyes, specific for cell membrane(PKH26and/or 67), cytoplasm (CFSE) and endo-lysoso-mal compartment (DiI) and stimulated with different apop-totic trigger, such as FasL, Trail and staurosporine, ableto trigger them towards an extrinsic or intrinsic pathway,respectively. Our results show that the triggers differentlypromote cell to cell communication. Firstly, the two celltypes show a different ability to form cell conjugates, animportant prerequisite for cell communication. Cell com-munication FasL-promoted mainly proceeds alongcytosolic material exchange via TNTs and MVs. Thebehaviour of Trail is similar to that one from FasL, even ifthe exchange is strongly reduced. On the contrary, stau-rosporine promotes in Jurkat cells mainly the exchange ofmembrane fragments. In order to further elucidate themechanism underlying this phenomenon we have evalu-ated the possible involvement of calcium, using specificchelators such as EDTA, EGTA and BAPTA-AM. Takentogether, our results suggest that the new routes of cellcommunication finely described in different biologicalcondition also occur during apoptosis, suggesting a keyrole in both life and death if the cell.

MINIMAL RESIDUAL DISEASE DETERMINATION ASA SURROGATE END-POINT IN ACUTE MYELOIDLEUKEMIAVenditti A.Cattedra di Ematologia, Dipartimento di Biomedicina ePrevenzione, Università Tor Vergata, Roma, Italyadriano.venditti@uniroma2.it

Cytogenetics and molecular abnormalities as assessedat diagnosis represent the most powerful prognosticatorsin acute myeloid leukemia (AML). However, since the out-come of patients allocated in the currently recognizedcytogenetic/genetic risk groups is highly variable, novelprognostic factors which take into account therapy andresponse related features are needed. Minimal residualdisease (MRD) might be one these new factors since itpotentially recapitulates all of the prognosticatorsinvolved in AML response to treatment such as cytoge-netics/genetics, multidrug resistance phenotype, pharma-cokinetic resistance and other still unknown factors. Anumber of study, using flow cytometry or molecular biolo-gy, have consistently demonstrated that MRD, whenpresent at any time-point after chemotherapy and stem

cell transplantation, is a significantly independent prog-nostic factors for survival estimates and duration of com-plete remission. Based on this, MRD detection is firmlyrising to the attention of the experts who envisage someunique implications potentially derived from its assess-ment: personalizing patient’s risk-assignment and thera-py, offering a more modern and realistic definition forcomplete remission and introducing a surrogate end-point for outcome of trials testing new agents in AML.Especially the potential role of MRD as a surrogate end-point is capturing the attention of the authorities such asthe Food & Drug Administration (FDA). In March 2013 atthe FDA headquarter, it was held a workshop focusingright on MRD detection as a surrogate end-point for clin-ical trials exploring new drugs in AML. A surrogate end-point is defined as a biomarker intended to substitute fora clinical efficacy endpoint; surrogate end-points areexpected to predict clinical benefit (or lack of benefit).Since MRD detection defines an end-point which occursprior to standard end-points such as overall survival ordisease free survival, it has the properties to serve as asurrogate end-point. In this view, the next challenge willbe to implement the procedures for MRD biomarker qual-ification in a way that correlation of MRD with traditionalend-points is strongly and convincingly established.

THE PARADIGM OF CHRONIC MYELOID LEUKEMIA:THE CRITICAL ROLE OF MRD MONITORING FORTHERAPEUTIC SUCCESS (AND CURE..?)Soverini S., Martinelli G.Department of Experimental, Diagnostic and SpecialtyMedicine, Institute of Hematology “L. e A. Seràgnoli”,University of Bologna, Italysimona.soverini@unibo.it

Chronic myeloid leukemia (CML) is invariably associatedwith the t(9;22) chromosomal translocation. This resultsin a BCR–ABL fusion gene encoding a tyrosine kinasewhose deregulation is both necessary and sufficient forthe initiation and maintenance of the disease. CML is oneof the first human malignancies for which the dream oftargeted therapy has come true with the advent of ima-tinib, the first tyrosine kinase inhibitor (TKI), in 2001. Untilthen, the deeper level of response patients could achievewith interferon alpha (in no more than 20% of the cases)was the complete cytogenetic response (CCyR), definedas the disappearance of metaphases positive for thet(9;22) translocation as assessed by chromosome band-ing analysis. Imatinib was shown to induce CCyR in morethan 80% of patients. It soon became evident that withimatinib, we could afford asking for more – that is, askingfor molecular responses. Molecular monitoring toolsallowing to qualitatevely detect the presence or absenceof the BCR-ABL fusion transcript were already available– they had been developed in an attempt to identify thoseCML patients who did not require further treatment afterallogeneic stem cell transplantation. Real-time reversetranscription (RT)-polymerase chain reaction (PCR)allowing to follow quantitatively the dynamics of BCR-ABL transcripts in blood and bone marrow, was thenintroduced in 1999. So, it was really a happy coincidencethat when imatinib entered clinical evaluation, this newand powerful tool had just been made available and couldthus complement the hematologic and cytogenetic follow-up of patients. Its high sensitivity soon made it the goldstandard to monitor the response of patients receivingTKI therapy. The application of real-time RT PCR indeedexcitingly showed that, as soon as CCyR is achieved, theleukemic burden continues to decline progressively over

time. As a consequence, two further steps beyond com-plete cytogenetic response were defined - MajorMolecular Response and Complete Molecular Response.Major Molecular Response (MMR) indicates a three logreduction in BCR-ABL transcript level with respect to thestandardized 100% baseline (hence, MMR=0.1% orlower). In a proportion of patients, however, we canobserve such a reduction in residual disease burden thatBCR-ABL becomes undetectable even by real-time RTPCR. This is what we call ‘CMR’ (Complete MolecularResponse; at least four log reduction i.e. 0.01% or lower).The achievement of specific molecular response mile-stones at specific timepoints has now entered the defini-tion of ‘optimal response’, ‘warning’ and ‘failure’. An opti-mal response is defined by BCR-ABL transcript levels≤10% at 3 months, ≤1% at 6 months, ≤0.1% at 12 monthsand ≤0.01% at 18 months of therapy. Second-generationTKIs (dasatinib, nilotinib) are now also available. Morepotent than imatinib, they are capable to induce higherMMR and CMR rates. The achievement of a stable CMRis the conditio-sine-qua-non for attempting treatment dis-continuation. This possibility, very attractive also from apharmacoeconomical point of view (TKIs are extremelyexpensive drugs), is now being explored in the context ofclinical trials whose preliminary results show its feasibili-ty at least in a proportion of patients. Supported byFondazione CARISBO, PRIN 2009 (prot. 2009JSMKY),AIL, AIRC.

MINIMAL RESIDUAL DISEASE STUDIES INCHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIAGaipa G.Pediatric Clinic, University of Milan Bicocca, M.TettamantiResearch Center, Monza, Italyg.gaipa@hsgerardo.org

Minimal Residual Disease (MRD) is a powerful and inde-pendent predictor of the overall response to treatment inChildhood Acute Lymphoblastic Leukaemia (ALL). Themost reliable and validated methods to assess MRD inALL are flow cytometric (FCM) analysis of leukemia-associated immunophenotypes and polymerase chainreaction (PCR) amplification of antigen-receptor generearrangements. However, a strong association betweenMRD and risk of relapse in childhood has been largelydemonstrated, irrespective of the methodology used.MRD is now used in several clinical trials for risk assign-ment and to guide clinical management. Low levels ofMRD at early time points may suggest to decrease treat-ment burden in patients who are likely to be cured withreduced intensity regimens. By contrast, high MRD levelsat late time points (such as end of consolidation) identifyALL subgroups which deserve investigation of moreeffective treatments. Recently, new MRD markers havebeen identified which can be incorporated to improve theapplicability and sensitivity of FCM-based MRD monitor-ing. The predictivity of MRD as a measurement of drugresponse in vivo may open new perspectives for its usein clinical decisions, and possibly as a surrogate for effi-cacy in the evaluation of novel therapeutic approaches.

FLOW CYTOMETRIC DETECTION OF ORAL PATHOGENBACTERIAManti A.1, Ciandrini E.2 , Baffone W.2, Campana R.2,Dominici S.2, Papa S.21Department of Earth, Life and Environmental Sciences.2Department of Biomolecular Science, University ofUrbino “Carlo Bo” Italyanita.manti@uniurb.it

Flow cytometry, in combination with fluorescent probetechniques, has been successfully applied for the rapidand specific detection and enumeration of bacteria. Itsuse has thus become an exceptionally powerful tool forinvestigating characteristic features of individual cells inheterogeneous populations.In particular, in this study, a flow cytometric approach wasused in order to detect four bacterial species (S. mutansATCC 25175, S. oralis ATCC 9811, F. nucleatum ATCC25586, P. gingivalis ATCC 33277) responsible of humanoral diseases. To this purpose, polyclonal antibodiesagainst these oral species were produced and tested forsensitivity and specificity.Results revealed high sensitivity for each antibodies. Thespecificity of each antibodies was tested towards otheroral bacterial species. Moreover, to reduce the eventualcross-reactivity, each polyclonal antibody was co-adsorbed with different mixtures of oral microorganisms.Furthermore, we tested some antimicrobial agents such ascarvacrol, a monoterpenic phenol with several medicinalproperties, and chlorhexidine, which has different biologi-cal properties, in order to investigate their antimicrobialactivity against S. mutans ATCC 25175, S. oralis ATCC9811, F. nucleatum ATCC 25586, P. gingivalis ATCC33277. Results evidenced that the exposure to carvacroland chlorhexidine for different times caused significanteffects on membrane integrity of all oral pathogens, sug-gesting that this essential oil may be useful for the preven-tion and treatment of periodontal diseases.In conclusion, the application of flow cytometry in combi-nation with specific polyclonal antibodies and some fluo-rochromes could be a helpful method for the analyses ofbacteria also in heterogeneous samples.

FLOWMOLECULARCYTOGENETICS: FLUORESCENCEIN SITU HYBRIDIZATION IN SUSPENSION (FISHIS)OFFERS NEWCHANCES TO FLOW SORTING OF CHRO-MOSOMESAND NUCLEILucretti S., Giorgi D., Grosso V., Farina A.ENEA, CASACCIA Research Center, Rome, Italysergio.lucretti@enea.it

Flow cytometry and cell sorting are techniques of growingrelevance for their analytical accuracy and high quantita-tive throughput which couple well with the powerful nextgeneration sequencing (NGS) technology which hasbeen opening new avenues in genomics. But large and/orpolyploid genomes are still a serious task to deciphersince the huge amounts of repetitive sequences, whichthey are made of largely, are still difficult to assemble in aproper and truthful way. Plants, and most of the industrialcrops such as wheat, cotton, banana, potato, coffee,have polyploid and/or large genomes which make thediscovery of the their DNA sequencing a daunting task.Therefore, the “chromosome approach”, or isolating chro-mosomes or chromosome arms via flow sorting accor-ding their different sizes, offers a clue to resolve such acomplexity by focusing sequencing to a discrete and self-consistent part of the whole genome. But the standardchromosome complement is not so polymorphic to permit

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a direct flow sorting of single type chromosomes and thisapproach is rather limited to very few plants where cyto-genetic mutants are available enabling enough chromo-some discrimination by size. We present a reliable, fastand cost effective method for Fluorescence In SituHybridization In Suspension (FISHIS) of plant chromo-somes that overcomes this limitation allowing chromo-some sorting based on total DNA content (size) but alsoon a FISH specific chromosome labeling pattern. FISHISrelies on alkaline DNA denaturation and on chromosomehybridization with readily available synthetic, fluorescent-ly labeled, repetitive sequences as probe. We show thatour method specifically labels and lets flow sorting of sev-eral chromosomes from standard plant karyotypes. Giventhe ubiquitous occurrence of used repetitive sequences,the abundance of their variants and their overall chromo-some-specific distribution, FISHIS experiments potential-ly open the access to virtually any complex eukaryoticgenomes and chromosomes.

TUMORMETASTASIS: EMT, STEMNESSANDMIGRATIONPirozzi G.Cancer Stem Cells Unit-National Cancer Institute-Naples, Italyg.pirozzi@istitutotumori.na.it

The value of cell sorting strategies for cancer researchstems from the tumor heterogeneity and inability of otherresearch approaches to resolve phenotypic and genomicdifferences in cancers. Solid tumors are composed of dif-ferent cell subpopulations which have different functionsin morphological, transcriptional, epigenetic and genetictraits. Flow cytometry activated sorting (FACS) allows forisolation and purification of cancer cells for subsequentanalysis (such as PCR, RNA or protein microarrays;Western blot, Southern blot, functional clonal assays),and highly selective enrichment for stem cell autologoustransplantation. Moreover, FACS made possible isolationof enriched cancer stem cells (CSCs), sorting of smallpopulations (and rare cells with frequency of 0.03%) forxenograft transplantation, and sorting single cancer cellsfor single-cell studies.Metastasis is the leading cause of death by cancer andremains poorly understood due to its complexity. It involvesmany steps: local invasion of cancer cells into the sur-rounding tissue, transport through the microvasculature ofthe lymph and blood systems, translocation, mainlythrough the bloodstream, to microvessels of distant tis-sues, exit from the bloodstream, survival and adaptation inthe distant microenvironment, and, finally, formation of asecondary tumour. Recent researches have demonstratedthat epithelial-to-mesenchymal transition (EMT) plays akey role in the early process of metastasis of cancer cells.Transforming growth factor-β1 (TGF-β1) is the majorinductor of EMT. In this context, aim of this study is to useFACS for isolation of CSCs as cells positive for CD133marker and Side Population (SP) and non-CSCs as cellsnegative for CD133 marker and SP, to treat them withTGF-β1 and, then, to investigate their role in migrationstarting from a A549 lung cancer cell line.To isolate CSCs and non-CSCs starting from the A549cell line, we sorted both for CD133 expression and SPparameters. We found that the mean expression levels ofCD133 were about 4%. The presence of SP cells in theA549 cell line was examined by staining cells withHoechst 33342 to generate a Hoechst blue–red profile.On the basis of the variance in Hoechst 33342 labeling,we gated the strongest dye efflux cells as SP+ cells,which were in the lower left quadrant of the FACS profile.As a control, the ATP-binding cassette (ABC) transporter

inhibitor verapamil was added to reduce the capacity toexclude Hoechst 33342. SP cells were detected in A549cells, with a mean percentage of about 15% of the totalcell population, and the SP cell fraction was abolished inthe presence of verapamil. The purity of sorted popula-tions was routinely 95% and sorted cells both for CD133and for SP were used for TGF-β1 treatment assay, RT-PCR, immunofluorescence and in vitro migration assays.We demonstrate that TGF-β1 induced EMT in both CSCand non-CSC A549 sublines, up-regulating the expres-sion of mesenchymal markers such as vimentin and Slug,and down-regulating levels of epithelial markers such ase-cadherin and cytokeratins. CSC and non-CSC A549sublines undergoing EMT showed a strong migration andstrong levels of MMP9 except for the CD133- cell frac-tion. OCT4 levels are strongly up-regulated in all cell frac-tions except CD133- cells. On the contrary, wound sizereveals that TGF-β1 enhances motility in wild-type A549as well as CD133+ and SP+ cells. For CD133- and SP-

cells, TGF-β1exposure does not change the motility.Finally, assessment of growth kinetics reveals majorcolony-forming efficiency in CD133+ A549 cells. In partic-ular, SP+ and SP- A549 cells show more efficiency to formcolonies than untreated corresponding cells, while forCD133- cells no change in colony number was observ-able after TGF-β1 exposure. We conclude that it is possi-ble to highlight different cell subpopulations with differentgrades of stemness: cells expressing CD133 could be atrue migrating subpopulation in lung adenocarcinomawhile SP+ cells could be considered the stationary CSCsubpopulation. Then, we hypothesize also that SP- cellfraction could represent a potential stem subpopulationused by tumours as a reservoir of stem cells in certainconditions.

INNATE IMMUNITY AND TUMOR PROGRESSION: AFLOW CYTOMETRIC POINT OF VIEW!Anselmo A., Germano G., Buracchi C. and Allavena P.Humanitas Clinical and Research Center, Department ofInflammation and Immunology, Rozzano, Italyachille.anselmo@humanitasresearch.it

The last decade witnessed an ever-growing awarenessof the promoting role of chronic inflammation in cancerinitiation and progression. Cancer-related inflammation isnow recognized as a hallmark of tumors. Tumor-associa-ted macrophages (TAM) and myeloid cells (monocytesand Myeloid-Derived Suppressor Cells) are present inlarge numbers in tumor tissues and are key promoters ofboth angiogenesis and suppression of adaptive immuneresponses. Moreover clinical and experimental evidencehas shown that high density of these leukocyte subsets isassociated with poor patient prognosis and resistance totherapies.Interestingly several studies have shown that tumor pro-gression can lead to extramedullary haematopoiesis thatcauses changes in leukocyte compositions in severalorgans like bone marrow, blood and spleen, while at thetumor site the increase of myelo-monocytic cells parallelswith necrosis due to the growth of the tumor mass. Thiscomplex framework requires the use of different antibo-dies for a correct flow cytometric analysis, in the presen-ce of dyes able to exclude dead cells. The immunophe-notypic analysis of myeloid cell subsets through flowcytometry both in humans and murine models by a multi-parametric approach involves the use of the right combi-nation of fluorophores in order to minimize the compen-sation and the aspecific signal derived from the high auto-fluorescence of these cells.

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Indeed setting up a reproducible and efficient method formyelo-monocytic cell detection through flow cytometryshould contribute to understand the complex cross-talkbetween the tumor and the immune system.In addition, the targeting of myeloid cells in tumors is con-sidered a promising therapeutic strategy: the depletion ofTAM or their “re-education” as anti-tumour effectors iscurrently under clinical investigation and hopefully willcontribute to the efficacy of conventional anti-cancer tre-atments.

CONVENTIONAL AND MULTISPECTRAL IMAGINGFLOW CYTOMETRY: INTEGRATED ANALYSIS OFAPOPTOSISDal Col J, and Dolcetti R.Cancer Bio-Immunotherapy Unit, Dept. of TranslationalResearch, C.R.O. - IRCCS, National Cancer Institute,Aviano, Italyjdalcol@cro.it

Apoptosis is a complex multi-step process. The assaymost commonly used to discriminate between viable anddead cells is based on the staining with Annexin-V and acell-impermeant nuclear dye. This analysis considersonly one of the hallmarks of apoptosis: the exposure ofphosphatidylserine on cell surface. However, there is theincreasing need to evaluate also other significant eventssuch as caspase activation, DNA fragmentation and thebalance between pro- and anti-apoptotic proteins.Multispectral Imaging Flow Cytometry (MIFC) combinesthe capabilities of microscopy and flow cytometry inte-grating the phenotypic data with information about celland nuclear morphology, protein localization and protein-protein interaction. Considering these peculiarities, werecently combined Conventional Flow Cytometry (CFC)and MIFC to better characterize the molecular mecha-nisms underlying the apoptotic effects of retinoic acid(RA) plus interferon-α (IFN-α) in mantle cell lymphoma.We demonstrated that the treatment triggers both intrin-sic and extrinsic apoptotic pathways and caused Bak andBax conformational chenges. We also showed thatRA/IFN-α induced the expression of Noxa and, takingadvantage of MIFC, we demonstrated that the upregula-tion of this protein was associated with the nucleartranslocation of the transcription factor FOXO3a. Finally,given the up-regulated Noxa co-immunoprecipitated withMcl-1 and Bfl-1, sequestering them from complexes withthe apoptotic effector Bid, we analyzed their interaction invivo. In details, we detected Mcl-1/Bid and Bfl-1/Bid co-localization in viable cells by MIFC, revealing that thismechanism is important to prevent Bid activation.In conclusion, our findings indicate that the integrationbetween CFC and MIFC may allow a more precise iden-tification of critical mediators of drug-induced apoptosis,leading to a more in-depth understanding of their regula-tion and interaction with specific partners of the complexapoptotic pathways.

MULTIPLE EFFECTS OF A SODIUM/HYDROGENANTIPORTER INHIBITORAredia F.,1 Giansanti V.,1 Mazzini G.,1,2 Savio M.,3Torriglia A.,4,5,6 Scovassi A.I.11Istituto di Genetica Molecolare CNR, Pavia, Italy;2Dipartimento di Biologia & Biotecnologie “L. Spallanzani”Università di Pavia, Italy; 3Dipartimento di MedicinaMolecolare, Università di Pavia, Italy; 4U872 eq. 17, Centrede Recherches des Cordeliers, INSERM, Paris, France;5Université Pierre et Marie Curie Paris 6, France;6Université Paris Descartes, Francearedia@igm.cnr.it

Amiloride derivatives are new promising chemotherapeu-tic agents. A representative member belonging to thisfamily is the sodium-hydrogen antiporter inhibitor HMA

Cell Cycle and Apoptosis

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(5-(N,N-hexamethylene amiloride), which induces cellularintracytosolic acidification and activates apoptosis. Wecharacterized the response to HMA of the SW613-B3colon carcinoma cell line that shows an intrinsic resist-ance to a panel of drugs, showing that HMA is able toinduce multiple effects. First, we detected that HMAimpairs cell survival in a dose dependent manner and inan irreversible way; then, we analyzed cell morphologyand we observed that cell number appear to be reducedin a dose-dependent manner and surviving cells werecondensed; also the cytoskeletal pattern was dramatical-ly altered. Thus, we pointed out the possible effects ofthis drug on DNA; we demonstrated that HMA is able toinduce alteration in cell cycle distribution, leading to aG2/M block; furthermore, DNA damage was evidenced bythe comet assay, which revealed the presence of a con-siderable amount of DNA damage, both as SSBs (singlestrand breaks) and oxidized pyrimidine or purine. Wefinally identified that in response to HMA, these cells acti-vate programmed cell deaths, which are independent ofcaspases activation (like parthanatos and LEI/L-DNase IIpathway). In addition, also autophagy occurs in ourexperimental system, thus supporting the idea that drugresponse is characterized by the firing of several deathpathways, which often are co-regulated.

PROLIFERATIVE EFFECT OF LOW-LEVEL LASERIRRADIATION ON BONE MARROW-DERIVEDMESENCHYMAL STEM CELLSBloise N.,1,2 Ceccarelli G.,2,3, Mantelli M.,6 Minzioni P.,4Mazzini G.,5 Avanzini M.A.,6 Cusella De Angelis M.G.,2,3

Imbriani M.,3,7 Visai L.1,2,7

1Dep. of Molecular Medicine, 2Ctr for Tissue Eng. (C.I.T.),3Dep.of Public Health, Exp. Medicine and Forensic, 4Dep.of Industrial and Information Eng., 5IGM-CNR and Dep. ofBiol. &,Bioteh. Univ. Pavia; 6Pediatric OncohematologyLab., Policlinico S.Matteo Foundation, IRCCS, Pavia;7Dep. of Occupational Medicine, Ergonomy andDisability, S. Maugeri Foundation, IRCCS, Pavia, Italynora.bloise@unipv.it

Low-level laser irradiation (LLLI) was found to increaseproliferation potential of various types of cells in vitro.However, the mechanisms associated with this phenom-enon remain poorly understood. Human bone marrow-derived mesenchymal stem cells (hBMSCs) have shownto be an appealing source for cell therapy and tissueengineering. In this study we examined the in vitro effectof a single or a multiple doses of LLLI on proliferation ofMSCs isolated from adult human bone marrow of differ-ent donors. The cells were irradiated with a red wave-length diode laser (659 nm) and exposed to differentlaser doses (0.5, 2, 5 J/cm²) for 1 day and 3 consecutivedays. Proliferation was evaluated using a quantitativeassay. Both single and multiple doses of LLLI enhancedthe proliferation of hBMSC isolated from all donors. A sig-nificantly cell growth increase was detected in groupsirradiated for three consecutive days compared to a sin-gle dose or control group. Colony Forming Unit-FibroblastExamination and Immunofluorescence Analysis of stemcells markers showed that laser treatment did not changetheir stemness characteristics. These preliminary resultsshowed that LLLI affected positively hBMSCs prolifera-tion in vitro and it may have an important impact for theuse of mesenchymal stem cells in regenerative medicine:in fact, LLLI may be use as a “photoceutical” bioreactorfor in vitro stem cells preconditioning prior to transplanta-tion. In this context, our final research aim is understandwhen and how physical stimuli can be useful for promot-

ing regeneration also for stem cells seeded and culturedon biomaterials.

EFFECTS ON NEUROBLASTOMA CELL CULTURESOF CISPLATIN AND PtAcacDMS (a new platinumcompound)1Dal Bo V., 1Piccolini V.M., 2Santin G., 1Grimaldi M.,1Barni S., 2Mazzini G., 1Veneroni P., 3De Pascali S.A.,3Fanizzi F.P., 1Bernocchi G., 1,2Bottone M.G.1Department of Biology and Biotecnology “L.Spallanzani”, University of Pavia, Italy; 2Institute ofMolecular Genetics of CNR, Pavia, Italy; 3General andInorganic Chemistry Laboratory, Department of Biologicaland Environmental Sciences and Technologies(DiSTeBA), University of Lecce, Italymazzi@igm.cnr.it

Cisplatin (cisPt) is one of the most active anticancer drugused in chemotherapy against several types of solidtumours. It binds DNA, forming adducts and blocking fun-damental cellular processes, such as transcription andtranslation. It has also recently been shown that it acts atcytoplasmic level, by damaging organelles and inducingcell death. Despite the great efficacy, cisPt has severalside effects, such as nephrotoxicity and ototoxicyty andinduces drug resistance, which greatly compromise itshealing potential. For this reason it became necessary tofind some new platinum-based drugs, which could beable to overcome these limits. A new platinum compound[Pt(O,O’-acac)(gamma-acac)(DMS)] (PtAcacDMS) wassynthesized; it reacts preferentially with protein thiol orthioether groups, suggesting that its targets are cytoplas-mic instead of nuclear. The aim of our research was tocompare the ability of cisPt and PtAcacDMS to drive celldeath in neuroblastoma cell cultures and to investigatethe intracellular mechanisms of in vitro cytotoxicityinduced by these platinum compounds. Our data showthat both cisPt and PtAcacDMS cause apoptosis by dif-ferent pathways: extrinsic (soluble molecules interactwith specific receptors on the cellular membrane trigger-ing death signal transduction) and intrinsic (triggered byintracellular stimuli or DNA damage), both inducing theactivation of some specific cytosolic proteases, the so-called caspases. These proteins have several targetsinside the cell, such as proteins and factors involved incell structure maintenance or enzymes controlling DNAmetabolism. In previous data we showed the higher toxicpotential of PtAcacDMS on cells respect cisPt: as a mat-ter of fact the apoptotic index is higher after PtAcacDMSadministration at a concentration four times lower thancisPt (10µM PtAcacDMS vs 40mM cisPt), suggesting thatthis new compound exerts a more relevant cytostaticaction. We also investigated the apoptosis/autophagyratio by treating cells for 48h with cisPt or PtAcacDMSand 7 days of recovery (drug-free medium), followed by a“replantation” in new flasks. Cells treated with cisPtexhibited autophagic characteristics, while cellsprocessed through the same treatments, but withPtAcacDMS, were apoptotic. In conclusion, cisPt inducesautophagy, a possible form of drug resistance, whilePtAcacDMS seems to lead tumour cells to death, show-ing antineoplastic effectiveness in vitro.

CELL CYCLE AND SURVIVAL PERTURBATIONSINDUCED BY NEUTRON AND GAMMA IRRADIATIONS:A COMPARATIVE METHODOLOGICAL STUDYFerrari C.,1 Cansolino L.,1,5 Clerici A.M.,1 Zonta C.,1Dionigi P.,1,5 Mazzini G.,2 Altieri S.,3,4 Ballarini F.,3,4

Bortolussi S.3,4, Postuma I.,3,4 Protti N.,3,4.1Dept. of Clinico-Surgical Sciences, Exp. Surg. Lab.;2IGM-CNR and Dept. of Biology and Biotechnologies “L.Spallanzani”, 3Dept. Physics; Univ. of Pavia; 4INFNPavia; 5IRCCS S. Matteo Hospital, Pavia, Italy.ferraric@unipv.it

Boron Neutron Capture Therapy (BNCT) is a non conven-tional binary radiation therapy for cancer treatment based onthe selective accumulation of 10B carriers in tumor followedby neutron irradiation. The highly ionizing particles, a-parti-cles and 7Li ions, generated by the neutron capture in 10B,dissipate their energy in a short range, comparable to thecell diameter. The radiation lethality is therefore confined tothe neoplastic boron enriched cells while the surroundinghealthy tissue is spared. This peculiarity makes BNCT aselective and effective therapeutic approach expecially incase of disseminated tumors.The present study was carried on in the frame of the pre-clinical in vitro validations aimed to verify the BNCT effi-cacy and applicability to liver or lung coloncarcinomametastases and to limb osteosarcoma. A comparative cellsurvival and cell cycle study was performed betweenadherent and suspended coloncarcinoma and osteosar-coma cell lines, in order to evaluate the alterationsinduced by thermal neutron and gamma irradiations. Incase of neutron irradiation analyses were performed onboron loaded and unloaded coloncarcinoma andosteosarcoma cell lines.Cell survival data, assessed by cloning assay, evidencedthat: i) a cell line-dependant radiosensitization can benoticed in case of adherent cells exposed to both radia-tion types, being the coloncarcinoma cell line the lessdamaged one; ii) adherent cells are much more sensitiveto radiations than suspended cell.Cell cycle cytofluorimetric DNA analyses showed an evi-dent dose dependant block in G2 phase for cells irradiatedin the suspended condition. Conversely, no evidence ofcell cycle delay was observed in case of adherent cells.The different behaviour between adherent and suspend-ed cells when exposed to radiations must be consideredin the evaluation and comparative studies of BNCTinduced damages.

FUNCTIONAL CHARACTERIZATION OF A VEGFMIMETIC PEPTIDE BY MEANS OF FLOW CYTOMETRYFesta L.,1 Celentano V.,1 Di Gaetano S.,2 D’Andrea L.D.,2Capasso D.31Diagnostica e Farmaceutica Molecolari Scarl, Napoli,Italy. 2Istituto di Biostrutture e Bioimmagini, CNR, Napoli,Italy. 3Centro Speciale per le Biotecnologie Federico II,Università degli studi di Napoli “Federico II” Italydomenica.capasso@unina.it

Angiogenesis consist of a series of interactive events thatinclude endothelial cell differentiation, proliferation andmigration, which lead to form new vessels. VEGFRs aretyrosine kinases receptors implicated in process of angio-genesis, focus of different study aimed at developing suit-able molecules for regulating angiogenesis.In previous studies, we designed, synthesized and char-acterized a peptide involved in receptor recognition, QKpeptide. This molecule reproduces the α-helix 17–25 ofthe VEGF binding interface. In vitro biological assays

suggested that QK shares numerous biological proper-ties of VEGF.Here we report the biological characterization of a QKanalogue peptide, named IQ. We demonstrated the spe-cific binding of peptide to VEGFR-2 by flow cytometry.Moreover, we showed that it is able to induce endothelialcell proliferation in a dose dependent manner and to res-cue cells from apoptosis. These findings indicate that IQplays a role on endothelial cell signalling, showing aVEGF-like behaviour and provides the basis for a ration-al design of a novel peptide-based drug for medical appli-cations in therapeutic angiogenesis.

A FLOW CYTOMETRIC ANALYSIS OF MITOCHONDRIALCELL DEATH IN HGFs/S. mitis CO-CULTURE MODELGallorini M., Sancilio S., Di Giulio M., Cataldi A.,De Colli M., di Giacomo V.Dipartimento di Farmacia, Università G. d’Annunzio,Chieti-Pescara, Italymarialucia.gallorini@unich.it

Resin monomers like TEGDMA (triethylene glycoldimethacrylate) enhance the formation of reactive oxy-gen species (ROS) in human gingival fibroblasts (HGFs)and this event seems to be associated with apoptosis.The acute depletion of mitochondrial GSH exerted byTEGDMA induces ROS production, collapse of the mito-chondrial membrane potential (MMP) and cell death. Ourwork aims to understand the mechanisms that underliemitochondrial apoptosis in HGFs co-cultured withStreptococcus mitis, a microorganism usually present inthe oral cavity, in the presence of saliva and treated with1 mM TEGDMA. Different flow cytometric analyses werecarried out: reactive oxygen species detection by CM-H2DCFDA (5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate, acetyl ester), TMRE (tetramethyl-rhodamine, ethyl ester) assay to label active mitochon-dria, Annexin V/PI detection of apoptotic and necroticcells and imaging flow cytometry analysis of BAX intracel-lular expression. Our results suggest that, in HGFs treat-ed with TEGDMA, the presence of S. mitis and/or salivareduces ROS production, BAX expression and apoptoticcell percentage while, on the other hand, increases MMPpotential. In the presence of N-acetylcysteine 10 mM, awell known non-enzymatic antioxidant, the apoptotic pop-ulation is reduced, mainly in the presence of saliva andbacteria. These results, shedding more light on the bio-logical and molecular events that occur upon TEGDMAtreatment in vitro in a co-culture model that mimics theenvironment of the oral cavity, award a key role to oralbacteria and saliva in preventing oxidative stress-inducedmitochondrial cell death in HGFs.

RAPAMYCIN (RAPA) PREVENTS LPS-INDUCEDENDOTHELIAL CELL DYSFUNCTIONGigante1 M, Stasi1 A, Castellano1 G, Intini1 A, Pontrelli1 P,Divella1 C, Grandaliano2 G and Gesualdo1 L.1Dept. of Emergency and Organ Transplantation,University of Bari, Italy. 2Dept. of Surgery and MedicalSciences, University of Foggia, Italy.gigantem@libero.it

Introduction and aims: Endothelial cells (ECs) activationhas a pivotal role in sepsis-induced AKI. Sepsis mayinduce phenotypic alteration of ECs by several mecha-nisms. The aim of our study was to evaluate the effects ofLPS on ECs and the role of RAPA in the modulation ofendothelial dysfunction.Methods: After 24h or 48 of LPS activation, ECs were

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analyzed by MTT cell viability assay and flow cytometryanalysis.Results: Through MTT assay, we observed a significantlyproliferation of ECs after LPS activation LPS 4�g/ml (p=0.04). When we addedd RAPA (5 nM) we found that the cellgrowth was restored to the basal condition by inhibiting LPS-induced activation (p = 0.05). Flow cytometry analysis (AnnV/ IP) showed that only a small percentage of ECs underwentto apoptosis after LPS activation (8.9% ±1.6 vs basal 5,9%± 1.2). Moreover, the addition of RAPA did not induce apop-tosis in LPS activated ECs. Interestingly, LPS-activated ECsshowed a reduction in endothelial markers such as CD31(66.46% ± 4.69 vs basal: 90.6% ± 4.12, p=0.003) and VE-cadherin (23.8% ± 5.24 vs basal: 54.4% ± 4.26, p=0.003)and an increased expression of fibroblast markers N-cad-herin (66.8% ± 4.84 vs basal: 8.46% ± 1.4, p=0.004),Vimentin (60.27% ± 7.44 vs basal: 40.69% ± 2.24, p=0.03)and FSP-1 (53,54% ± 6.81 vs basal: 26.47% ± 5.05,p=0.04). Interestingly, the addition of RAPA abrogated theLPS-induced endothelial dysfunction by restoring theexpression of CD31 and VE-cadherin, and inhibiting theexpression of de-differentiation markers such as N-cadherin,Vimentin and FSP-1.Conclusions: These data suggest that LPS acts directlyon ECs by activating cellular proliferation and promotingendothelial dysfunction and de-differentiation. The inhibi-tion of ECs activation via RAPA may represent a possibletherapeutic strategy to prevent endothelial dysfunctionduring sepsis-induced AKI.

LEPTIN INDUCES THE GENERATION OF MICROPAR-TICLES BY HUMAN MONONUCLEAR CELLSPetrini S.1, Insalco D.1, Neri T.1, Lombardi S.2, Balia C.1,Scalise V.1, Cianchetti S.1, Pedrinelli R.1,3, Celi A.11Laboratorio di biologia cellulare e respiratoria, Dipartimentodi patologia chirurgica, medica, molecolare e dell’area criti-ca, Università di Pisa, Italy. 2SSD Immunologia, Allergologiae Patologia Molecolare, USL1, Massa e Carrara, Italy;3Istituto Nazionale di Ricerche Cardiovascolari, INRC,Bologna, Italy.silvia.petrini@for.unipi.it

Purpose: Microparticles (MP) are phospholipid vesiclesshed by cells upon activation or during apoptosis. MP areinvolved in numerous physiological processes, includingcoagulation and inflammation. Leptin, synthesized by adi-pose tissue, has been implicated in the regulation ofinflammation and the pathogenesis of thrombosis.Aim: Aim of our study is to test the hypothesis that one ofthe mechanisms of leptin is linked to the induction of pro-coagulant MP by human mononuclear cells (HMCs) andto investigate the intracellular mechanisms leading to MPrelease upon incubation with leptin.Methods: HMCs are isolated from the peripheral blood ofhealthy donors. Cells are incubated with leptin (10 µg/mL:4 hours). Leptin-stimulated cells are also incubated witha phospholipase C inhibitor, U73122 or with three differ-ent inhibitors of MAP kinases. MP generation is assessedas phosphatidylserine (PS) concentration by a prothrom-binase assay, after capturing the MP onto annexin V-coated wells and by cytofluorimentric analysis. HMCsderived MP were discriminated first by size, as eventsconforming to a light scatter distribution within the 0.5-0.9µm bead range in a SSC vs. FSC window and furtheridentified as CD14 and annexin V positive events afterincubation with fluorescein isothyocianate (FITC)-annex-in V and allophycocyanin (APC)-anti CD14 Ab, in a APCvs. FITC window.Results: Leptin significantly stimulates MP generation by

HMCs (0.38±0.12 vs 0.21±0.11 nM PS; mean±SEM).These results are confirmed by cytofluorimetric analysis(262±51.39 vs 38.67±10.09 events CD14+/annexinV+).U73122, 1µM, significantly inhibits leptin-induced MPrelease (0.32±0.15 vs 0.46±0.22 nM PS). MAPKinhibitors have no effect.Conclusions: Leptin induces the release of MP with a pro-coagulant potential by HMCs through a mechanism thatinvolves phospholipase C activation. These data are con-sistent with a role of leptin-induced procoagulant MPshed by MNCs in vascular diseases linked to obesity.

RUBINSTEIN-TAYBI SYNDROME LYMPHOBLASTOIDCELLS SHOW INCREASED SENSITIVITY TO OXIDATIVEDNADAMAGETillhon M.,1 Dutto I.,1 Quaranta V.,1 Gervasini C.,2

Larizza L.2 and Prosperi E.1

1Institute of Molecular Genetics (IGM), CNR, ViaAbbiategrasso 207, Pavia, Italy; 2Medical Genetics,Department of Health Sciences, University of Milano,Milano, Italytillhon@igm.cnr.it

The Rubinstein–Taybi syndrome (RSTS) is a genetic dis-order associated with defects in postnatal growth, micro-cephaly, intellectual disability and increased risk oftumors. The exact molecular etiology of RSTS is notclearly understood; however it is widely accepted thatRSTS is associated with mutations and chromosomalrearrangements (microdeletions and translocations) withbreakpoints at 16p13.3, where the gene encoding thecAMP Response Element-Binding Protein (CREBBP orCBP) is located. About 60% of RSTS individuals carry aheterozygous mutation/deletion of CREBBP, while asmall percentage (~3%) of RSTS are caused by muta-tions in the EP300 gene (located at 22q13.2) encodingfor p300. CREBBP and p300 are highly homologous pro-teins with lysine acetyl transferase (KAT) activity, andplay a key role in transcription regulation. However, theyhave also distinct cellular functions and cannot alwaysreplace one another. Both proteins are involved in otheraspects of DNA metabolism, including cell cycle check-points (by acetylating p53), and DNA repair (by acetylat-ing factors involved in nucleotide (NER) and base exci-sion repair (BER) mechanisms, such as XPG, FEN-1,DNA polymerase β (pol β), some DNA glycosylases (e.g.TDG, NEIL2, OGG1), as well as PARP-1, and WRN andRECQL4 helicases. In agreement with these findings, wehave recently shown that depletion of both CBP and p300by RNA interference, significantly impairs NER. Thus,deficiency in CBP and/or p300 may result in genomeinstability and cancerogenesis, thereby explaining, atleast in part, the higher incidence of malignanciesobserved in RSTS patients. In order to study the involve-ment of CBP and p300 in response to DNA damage, andin particular in DNA repair, we have analyzed lym-phoblastoid cell lines derived from RSTS patients carry-ing monoallelic deletion, or mutations of CREBBP gene.Flow cytometry and BrdU labeling has been used toassess proliferative characteristics of these cell lines.Western blotting analysis has shown, as expected, areduced expression of CBP, and in one case also of p300protein in RSTS lymphoblasts, as compared to the lym-phoblastoid cell lines from normal donors. No significantdifferences were observed in the protein expression ofPCNA, and other related DNA repair proteins, such as XPproteins (A-G), PARP-1, XRCC1 and DNA pol β. Despitea similar proliferation rate, RSTS cells showed signs ofhistone H2AX phosphorylation, suggesting the presence

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AN INTEGRATED APPROACH TO UNDERSTAND THEMICROBIAL COMMUNITY STRUCTURE AND FUNC-TIONING IN COASTAL AREASBalestra C. and Casotti R.Stazione Zoologica A. Dohrn, Laboratory of FunctionalEcology and Evolution, Naples, Italyraffaella.casotti@szn.it

In November 2010, 32 stations were sampled to estimateheterotrophic bacteria and picophytoplankton abundanceby flow cytometry, bacterial community composition andmetabolic activity by CARD-FISH and MAR-CARD-FISH.Prochlorococcus was the most abundant autotrophicpicoplankter (1.88 ± SD 1.10x104 cell ml-1), followed bySynechococcus (1.50 ± SD 1.07x104 cell ml-1) andpicoeukaryotes (7.59± SD 6.20 x102cell ml-1). Theseshowed the highest concentrations in the Sele river estu-ary at surface (1.98x104 cell ml-1,4.89x104 cell ml-1,3.31x103 cell ml-1, respectively). Heterotrophic bacteriawere 6.01 ± SD 3.88 x105cell ml-1. Their concentrationsvaried from 1.48x105 and 2.17x106 cell ml-1 following thecoastal offshore gradient with higher concentrations nearthe rivers confirming their role as remineralizers of organ-ic matter and nutrient recyclers. Bacterial communitycomposition was analysed by CARD-FISH. 75% ± of totalcell counts were detected with the EUB 338 probe. Theoverall most abundant group was Alphaproteobacteria56% ± 6.7. The second group in terms of abundance wasCytophaga-Flavobacterium-Bacteroides 30% ± 5.4.Gammaproteobacteria were on average 22% ± 5.0.Along the Sarno and the Volturno river Cytophaga andGammaproteobacteria showed an increase from the off-shore stations (22% ± 2.1 and 19% ±1.8 CFB andGamma, respectively) to the coastal ones (30% ±1 and24% ± 2 CFB and Gamma respectively). On the contraryAlphaproteobacteria showed an opposite trend with high-er concentration offshore (55% ± 2.0) and lower near thecoast (44% ± 3.2). Among the Alphaproteobacteria,SAR11 was the most abundant clade (up to 30% of totalEubacteria counts). The least abundant clade at all sta-tions was Roseobacter (9% ± 3.4). These data are to becomplemented with information from two metagenomicsequences with the aim of providing integrated tools forthe assessment of biological properties of coastal areas.

EFFECT OF ALGAL POLYUNSATURATED ALDHEY-DES ON A NATURAL MARINE BACTERIAL COMMU-NITY FROM THE GULF OF NAPLESBalestra C., Galasso C., Casotti R.Stazione Zoologica A. Dohrn, Laboratory of FunctionalEcology and Evolution, Naples, Italyraffaella.casotti@szn.it

Polyunsaturated aldehydes (PUA) are algal secondarymetabolites toxic for different marine organisms. Theeffect of different PUA on community composition andactivity was tested on natural heterotrophic bacterialcommunities in July 2009. Triplicate surface sampleswere incubated with heptadienal (HEPTA), octadienal(OCTA), decadienal (DECA) and a mix of the last two(MIX) and assayed after 6 and 24 h. Initial bacterial abun-dances, estimated by flow cytometry, were 8.98 x 105 ±SD 2 x 105 cell ml-1 and did not vary significantly during

Environmental Sciencesand Toxicology

of endogenous DNA damage. In addition, all RSTS celllines tested were more sensitive to treatment with theoxidative agent KBrO3. Experiments have been per-formed to assess the recruitment of XRCC1 and DNA polβ to DNA damage sites, and the efficiency of DNA repairis being analysed by the Comet test. Preliminary resultssuggest a different and delayed kinetics in the repair ofoxidative lesions in RSTS cells; however, further investi-gations are required in order to fully characterize thebehaviour of these cell lines.

the experiment, probably due to a compensatory effect.Community composition and activity was estimated byCARD-FISH and MAR-CARD-FISH. After 6 h incubation,Gammaproteobacteria (Gamma), CFB and ROS cladeswere not affected by PUAs and even were stimulated dif-ferentially by some of the PUAs tested (up to 59% forGamma with MIX). Alphaproteobacteria (Alpha) were themost affected, with a dramatic decrease of 83%, whileSAR11 was not affected. The stimulatory effect was con-firmed after 24 h for Gamma and CFB, but not for ROS.Alpha recovered and even increased their contributiondespite the fact that the control had lowered in percent-age. At the end of the experiment, the community compo-sition had changed and mostly profited Gamma whichincreased their relative contribution from 19% to 38% rel-ative to the total Eubacteria which increased, in turn, from85% to 100%. The same was reflected by the MAR-CARD-FISH data, with Gamma, CFB and ROS showinga higher activity when compared to the t0 and the relativecontrol after 6 h exposure, especially with the MIX, whileAlpha were initially affected but recovered after 24 h. Themetabolic activity of SAR 11 was not affected by anytreatment. These data confirm previous results whereGamma was also the least affected by PUA and the MIXhad a stronger positive or negative effect than the singlePUA, suggesting a synergistic effect. The role of PUA inshaping the bacterial community is confirmed, with differ-ences depending from factors yet to be elucidated.

DISTRIBUTION AND COMPOSITION OF NATURALBACTERIAL COMMUNITY AT A LONG TERMECOLOGICAL RESEARCH STATION (LTER) IN THEGULF OF NAPLESBalestra C., Trano A.C., Casotti R.Stazione Zoologica A. Dohrn, Villa Comunale 80121,Napoli, Italycecilia.balestra@szn.it

Bacteria are important components of pelagic food webboth in terms of biomass and activity. Their abundance,distribution and taxonomic composition were studied dur-ing 2011 at the Long Term Ecological Research StationMareChiara, in the Gulf of Naples. Bacterial abundancewas estimated by flow cytometry and community compo-sition by CARD-FISH. Heterotrophic bacteria were homo-geneously distributed along the water column during win-ter as a consequence of seasonal mixing. The highestconcentrations were recorded in spring (2.72 x 106 cellml-1 in May), as well as in summer and autumn (2.70 x106 cell ml-1 and 2.17 x 106 cell ml-1 in August andNovember, respectively). These peaks followed chloro-phyll a and phytoplankton abundance distribution, likelyto indicate that the organic matter released by phyto-plankton was being utilized for bacterial growth.71% (±4%) of heterotrophic bacteria were detected usingthe Eubacteria probe by CARD-FISH. Among these, themost abundant group was Alphaproteobacteria (50%±5)with a peak in March (59% ±13%). This group is known toremineralize nutrients used by phytoplankton, which wellcorrelates with the abundance and chl a data. Within theAlphaproteobacteria, SAR11 was the most abundantgroup along the year, showing an opposite seasonaltrend to Roseobacter, which was generally found in lowerproportions. During the year Bacteroidetes andGammaproteobacteria were on average 22% (±5%) and21% (±6%), respectively. Bacteroidetes were the mostabundant group in April, maybe due to the lower watertemperature. This is possibly due to the fact that this bac-terial group is more efficient in using different organic

compounds at low temperatures in coastal waters. On thecontrary, Gammaproteobacteria increased up to 50 %during February due to external input. Altogether, thesedata indicate that, at the LTER MareChiara, bacterio-plankton abundance and composition depend on differentenvironmental factors, including temperature, salinity,nutrients and chlorophyll a.

DETECTING SPERM DNA FRAGMENTATION TODISCRIMINATE BETWEEN FERTILE AND INFERTILEMENCambi M.1, Natali I.2, Olivito B.3, Azzari C.3, Forti G.1,Baldi E.1 Muratori M.11Dept. of Experimental and Clinical Biomedical SciencesAndrology Unit, University of Florence, viale Pieraccini6,I-50139 Florence. Italy . 2Sterility Center, Obstetric andGynecology Unit, S.S. Cosma and Damiano Hospital,Pescia; 3Department of Health Sciences, A. MeyerChildren’s Hospital, Florence, Italymonica.muratori@unifi.it

Sperm DNA Fragmentation (sDF) is an anomaly of spermgenome consisting in single and double stranded DNAbreaks. The impact of sDF on reproductive outcomesremains elusive due to the conflicting results of clinicalstudies. The ability of tests detecting sDF to predict theoutcomes of reproduction is affected by many variables,including the sperm population where the damage isrevealed. Using TUNEL/PI, coupling the detection of sDFto the nuclear staining with propidium iodide, PI, ourgroup unveiled two flow cytometric sperm populationsthat differ for PI staining (termed PI brighter and PI dim-mer populations), for the amount of sDF and for cell via-bility. Indeed, PI dimmer sperm are all DNA fragmentedand not viable. Conversely, PI brighter sperm are bothfragmented and not fragmented and both viable and notviable. Based on this finding we reasoned that PI dimmersperm have no chance to participate in fertilization, thatthe fraction of sDF really impacting on reproduction is thatof PI brighter sperm and, within it, that of viable gametes.To verify this hypothesis, we set up a method able todetect sDF in viable spermatozoa by using a LIVE/DEADfixable stain that labels dead cells permanently, thusremaining even after processing samples by TUNEL forsDF detection. Then we compared the levels of sDF asmeasured in total, PI brighter and live spermatozoa in 23fertile and 22 infertile men. As expected, we found thatsDF resulted increased (p<0.05) in infertile respect to fer-tile subjects, both in total (44.6±18.8 vs 35.2±13.6%) andPI brighter (33.2±15.8 vs 24.3±10.8%) and live sperm(25.1±19.3 vs 13.6±6.2%). However, the percentageincrease in infertile vs fertile subjects was much greaterfor viable sperm (84.4%) respect to PI brighter sperm(36.4%) and total population (26.5%). In conclusion, theability of sDF to discriminate between fertile and infertilemen, ameliorates considering PI brighter and above allviable sperm, respect to total sperm population.

NANOFIBRILLAR CELLULOSE INDUCES CYTOTOXICEFFECTS IN HUMAN LEUKOCYTESCanonico B.,1 Manti A.,1 Boi P.,1 Arcangeletti M.,2 Gatti A.,3Papa S.21Department of Earth, Life and Environmental Sciences,University of Urbino “Carlo Bo”, Urbino, 2Department ofBiomolecular Science, University of Urbino “Carlo Bo”,Urbino, 3Nanodiagnostics srl and Council of Research ofItaly- ISTEC, Faenza (RA) Italybarbara.canonico@uniurb.it

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Nanofibrillar-cellulose (NFC) based materials haveattracted a growing interest in different fields of applica-tions such as packaging, adhesives, fibers, opto-elec-tronic and medical devices, and automotives componentsfor their innovative properties. For some alimentary andmedical use it is mandatory to verify its biocompatibility,namely its biological acceptability. The basic nanosafetytests verify if a material, its extracts or suspensionsinduce cell death, malformation, degeneration and lysis.To check possible cytotoxic effects, flow cytometric tech-niques and Electron Scanning Microscopic (ESEM)observations were applied to investigate toxic effects onhuman leukocytes (lymphocytes and granulocytes) afterNFC exposure.Different NFC samples called CTRL, KJ, TEMPO wereadministrated to the cells at different times (T=6h, 24h,48, 72h) and analysed by flow cytometry in order to eval-uate cell death (propidium iodide uptake), membranepotential (TMRE), mitochondrial cardiolipine content(NAO) and acidic compartment (AO).Results show a rise in apoptotic and necrotic cell numberespecially on granulocytes, for CTRL at 6h and 24h; whileTEMPO treatment induces a progressively increasing celldeath during all time course.ESEM observations confirm the cytometric data, espe-cially for the reduction of the cell number. focusing flowcytometric approach as an extremely appropriate anduseful tool to detect cytotoxic effects.Our results indicate that NFC suspensions (mainly CTRLand TEMPO samples) are proficient cytotoxic inducers, inboth number of cells involved and in degree of damageinduction, suggesting that these nanostructured materialsare probably not suitable to be used for food packagingand medical devices.

CYTO-GENOTOXICITY AND INFLAMMATORYRESPONSE IN HUMAN ALVEOLAR (A549) CELLSEXPOSED TO TITANIUM DIOXIDE NANOPARTICLESCavallo D., Ursini C.L., Fresegna A. M., Ciervo A., Maiello R.,Tassone P., Iavicoli S.INAIL - Italian Workers’ Compensation Authority –Research Area, Department of Occupational Medicine,Rome, Italyd.cavallo@inail.it

The increasing use of titanium dioxide nanoparticles(TiO2NPs) in consumer products (cosmetics and foodadditives) and industrial and medical applications high-lights the importance of understanding its potential toxiceffects. Since the respiratory tract represents an impor-tant exposure route for workers employed in the TiO2NPsproduction, we investigated the cyto-genotoxic andinflammatory effects induced by commercial TiO2NPs inhuman alveolar cells (A549) exposed to 1, 5, 10, 20 and40 µg/mL. Cytotoxic effects were studied by viabilityanalysis with WST1 test after 24h exposure and cellmembrane damage by LDH release assay after 30 min, 2and 24h. Direct/oxidative DNA damage was assessed byFpg-modified comet assay and the inflammatory potentialby IL-6, IL-8 and TNFα release by ELISA, both after 2and 24h exposure. We did not find significant cell viabili-ty reduction. Slight membrane damage was detectedafter 30 min and 2h at 40µg/mL. Slight direct DNA dam-age was found only after 2h exposure at the highest con-centration. A moderate induction of oxidative DNA dam-age was found at 40 µg/mL after 2h and at 5 µg/mL after24h exposure. A slight increase of IL-6 release wasdetected only at 5 µg/mL after 2h exposure. No effects onIL-8 and TNFα releases were observed. The results show

a lack of cytotoxic effects in terms of cell viability reduc-tion and a slight early membrane damage. No significantdirect DNA damage was also found while a slight oxida-tive DNA damage and IL-6 release was induced. Thesecretion of pro-inflammatory cytokine IL-6, involved inearly acute inflammatory response, and the slight oxida-tive DNA damage indicate the promotion of mild oxida-tive-inflammatory effects for this kind of NP on humanalveolar cells. Overall the findings confirm the low cyto-genotoxicity of TiO2NPs and an early inflammatoryresponse of A549 cells, demonstrating the suitability ofthis experimental system to study nanomaterial-mediatedtoxicity.

A FLOW MOLECULAR CYTOGENETIC APPROACHTO MAKE SIMPLER COMPLEX GENOME GENOMICS:FISHISGiorgi D., Farina A., Grosso V., Lucretti S.ENEA, C. R. CASACCIA, Unità Sviluppo Sostenibile edInnovazione del Sistema Agroindustriale - UTAGRI INN,Via Anguillarese 301, 00123 Roma, Italysergio.lucretti@enea.it

Genome complexity of major crop plants, which resultsfrom polyploidy and high abundance of non-genic repeti-tive sequences, has hampered a fast progress in theexploitation of genomic technologies as analytical andeven breeding tools. By enabling genome dissection intosingle chromosomes or chromosome arms via flow sort-ing, the ‘chromosome approach’ can contribute to reducegenome complexity. Up till now, this approach is restrict-ed to species, or special cytogenetic stocks, containingchromosome types that differ in size from the standardcomplement. The chromosome approach proved to bevery effective in dividing the large bread wheat genome insmall and significant parts, but it has in itself the limitationof being available in the genetic background of a specificvariety (e.g. T. aestivum Chinese Spring), not one ofmajor interest. Moreover, such an approach is unpracticalfor the most of plants and animals, precluding an easydevelopment of genome sequencing and assembling inthe case of complex genomes. A new method has beendeveloped which overcomes these limitations, termedFISHIS ‘‘Fluorescence In situ Hybridization InSuspension’’, allowing the coupling of the high discrimi-natory capabilities of FISH labeling and the high-through-put of flow-cytometry analysis (FCM) and flow-sorting toisolate pure chromosome and nuclei fractions, regardlessof the availability of cytogenetic stocks. FISHIS is a wash-less method that relies on readily available fluorescentlylabeled DNA repetitive sequences (e.g. SSRs) andemploys alkaline DNA denaturation to overcome all stepscausing chromosome clumping and losses during classicFISH. FCM-FISHIS can discriminate, and flow sort: i) theentire chromosome set of a single genome from thehomeologous ones in polyploid pasta and bread wheat; ii)several single-type chromosomes in a number ofTriticeae, and iii) the entire chromosome complement ofthe wild grain mosquitograss (D. Villosum L.). The FISHISmethod potentially extends the analytical and preparativepower of flow-sorting to each individual chromosome andnucleus of virtually any eukaryotic species, providing thatsuitable DNA probes and high quality chromosome sus-pensions would be both available.

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AN INTEGRATED FLOW CYTOMETRY-BASEDAPPROCH TO SHAPE NEW BEAUTY IN ORCHIDSGrosso V.*, Nardi L.**, Farina A*, Aracri B.***, Pashkoulov D.***Debora G.*, Lucretti S.**ENEA, C. R. CASACCIA, Unità Sviluppo Sostenibile edInnovazione del Sistema Agroindustriale - UTAGRI INN,Via Anguillarese 301, 00123 Roma, Italy. **ENEA CentroRicerche Casaccia, Unità Tecnica RAD, UTRAD FARM,Via Anguillarese 301, 00123 Roma, Italy.***FLORAMIATA, Località Casa del Corto, 53025Piancastagnaio (SI) Italysergio.lucretti@enea.it

Orchids account for several thousands of species, actu-ally more than 25,000 species, and have a consistentworldwide market which depends largely on the constantavailability of new and surprising varieties satisfying theconsumers’ request for. Therefore, breeding for new vari-eties and hybrid combinations attractive to the con-sumers is the main target in orchids. A main constraint toa classical breeding in orchids is their slow growth habitwhich may delay flowering up to five or more years fromthe crossing. A possible way to introduce new variabilityis to double the ploidy level of the wild and commercialvarieties; and polyploidy is a well known event which cancontribute in ornamental plant breeding. In orchids, poly-ploids showed larger flowers, extended blooming timeand multiple flowering. We have developed inDendrobium a highly regenerative tissue culture systemwhich makes use of PLBs (protocorm like bodies) in liq-uid culture to obtain a fast growing material to be utilizedfor polyploidization experiments. Polyploids may be gen-erated through the use of spindle-formation inhibitorswithout affecting cell-viability, if a proper concentration ofblocking agent would be supplemented into the culturesubstrate. A number of substrates both liquid and solid,were tested combining several spindle inhibitors(COLchicine and APM) with different concentrations andincubation timings. The fresh-weight changes, the regen-eration frequency and the ploidy evaluation were allassessed on the PLBs cultures highlighting the good per-formance of COL as a polyploid agent. Similar effortswere performed on Phalaenopsis e Cattleya, but withminor results. Ploidy early screening was carried out withflow cytometry (FCM) on nuclei extracted from 10-20 mil-ligrams of plant tissues, both PLBs or young leaftlets,allowing a clear discrimination of the ploidy levels afterfew weeks of in vitro subculturing. Regenerants with dif-ferent ploidy levels underwent to a molecular characteri-zation with RAPD and SSR showing their different genet-ic constitution, which was corroborated also by a fastergrowth, in respect to parentals. A genome-downsizingeffect was detected by FCM on several regeneratedplantlets since this phenomenon can’t be assessed easi-ly by standard cytological observation because of thevery tiny size of orchid chromosomes. More than 30 newDendrobium polyploid plantlets were identified andselected after no more than three months of in vitro sub-culturing, allowing a remarkable reduction in working-time and reagent-costs eventually needed to maintain alarge experimental population of orchid plants. This workis supported by MIPAAF, Project NOVAORCHID(D.M.11074/7643/09).

FLOW CYTOMETRIC EVALUATION OF INTRACELLULARUPTAKE AND TOXICITY IN LYMPHOCYTES ANDMONOCYTES EXPOSED TO NANO OR MICRO SIZEDTiO2 PARTICLESLeter G.,1 Zijno A.,2 Andreoli C.,2 De Berardis B.,2 Barone F.,2Degan P.,3 Pacchierotti F.11Laboratory of Toxicology, ENEA Casaccia, Rome; 2DeptEnvironment and Primary Prevention, Istituto Superioredi Sanità, Roma; 3Molecular Mutagenesis and DNARepair, Istituto Nazionale per la Ricerca sul Cancro,Genova Italy,giorgio.leter@enea.it

Increasing scientific interest on nanoparticles applicationis driven by their unique physicochemical properties asreported in several studies carried out in the last fewdecades. A multiplicity of nanomaterials of variousshapes have been engineered for technological improve-ment in many branches of medicine, electronics as wellas generic consumer goods. In parallel, a growing con-cern about possible health risks associated to nanoparti-cle exposure has emerged and scientific investigation arein progress. At a cellular level, adverse effects may stemfrom intracellular uptake of nanoparticles and/or indirectstress response mechanisms.We analyzed by flow cytometry (FCM), reactive oxygenspecies (ROS) production and cell viability in immunola-belled subpopulations of human lymphocytes and mono-cytes exposed to nano or micro sized TiO2 particles.DNA damage evaluated by Comet assay and 8-oxoGincreased levels were observed in unstimulated lympho-cytes. Conversely no micronucleus induction wasobserved in stimulated lymphocytes.We showed that different levels of toxicity in the two celltypes might be directly correlated with different levels ofparticle uptake measured by FCM scatter signals. Inaddition, the lower level of uptake in monocytes exposedat 4 °C than in monocytes exposed at room temperaturesuggested that in these cells uptake occurred by anactive endocytosis mechanism.

AN IMPROVED FLOW CYTOMETRIC (FCM)IMMUNODETECTION METHOD TO EVALUATE SPERMDNA GLOBAL METHYLATION LEVEL (DGML)Leter G1, Eleuteri P1, Pacchierotti F1, Bonde JP2, Toft G3,Jönsson BAG4, Giwercman A5, Pedersen HP6, Ludwicki JK7,Zviezdai V8, Heederik D9, and 1Spanò M.1Laboratory of Toxicology, ENEA Casaccia, Rome, Italy;2Bispebjerg University Hospital, Copenhagen, Denmark;3Danish Ramazzini Center, Aarhus University Hospital,Aarhus, Denmark; 4Department of Laboratory Medicine,Lund University, Lund, Sweden; 5Reproductive MedicineCentre, Skåne University Hospital, Malmö, Sweden;6Centre for Arctic Environmental Medicine, Nuuk,Greenland; 7National Institute of Public Health - NationalInstitute of Hygiene, Warsaw, Poland; 8Kharkiv NationalMedical University, Kharkiv, Ukraine; 9Institute for RiskAssessment Sciences, Utrecht University, Utrecht, TheNetherlandsgiorgio.leter@enea.it

DNA methylation is an epigenetic mechanism involved inthe regulation of parental imprinting, gene expression,and genomic stability. Aberrant DNA methylation hasbeen associated with aging, diseases and exposure toenvironmental contaminants potentially toxic for humanmale reproduction. 5-methyl Cytosine (5-mC) quantitativeimmunostaining is a cheap method for globally assessingsperm DNA methylation, but the pertinent literature is

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scanty and lacks information about quality control andreproducibility of the fluorescence measurements. In theframe of a EU project aimed at studying how contami-nants may impact on male reproductive disorders, weoptimized and applied a FCM method for 5-mC immun-odetection on sperm. The probe consisted of an anti-5-mC mouse monoclonal antibody coupled withAlexaFluor488-conjugated Fab anti-mouse IgG1 frag-ments. The sperm DGML was evaluated in deep frozenarchived samples from 270 healthy fertile men fromGreenland, Ukraine, and Poland. Variability of themethodology, evaluated with the help of a reference sam-ple, resulted <15%. Even though this procedure does notstoichiometrically determine the number of 5-mCs in thegenome, the green fluorescence intensity mirrors thenumber of 5-mCs accessible to the antibodies’ complex.Sperm DMGL resulted quite independent from conven-tional semen quality parameters. Partially funded byProject CLEAR “Climate, Environmental Contaminantsand Reproductive Health” (www.inuendo.dk/clear),European Commission 7th Framework Programme FP7-ENV-2008-1 Environment (including Climate Change)Grant no. 226217.

THE SIGNIFICANCE OF GENETIC DETERMINANTSOF DETOXIFICATION AND ENVIRONMENTAL RISK-FACTORS IN CHRONIC DISEASES AND CANCERPREVENTIONMaggi Herbring F.1, Schollenberger L.1, Jennrich P.2,Herbring V.31FraMaGENE, Heidelberg, Germany. 2PRAXISKLINIK,Wuerzburg, Germany. 3Technische UniversitätDarmstadt, Germanyfm@framagene.com, peter_jennrich@yahoo.de,v.herbring@hotmail.de

Several exogenous and endogenous factors, includinginherited differences in metabolic activity, environmentalexposures to genotoxic agents, and nutritional habitscause individual predisposition to cancer and chronicdiseases.The cellular mechanisms for detoxification, working prin-cipally in two phases, are indispensable to protect cellsfrom damage caused by exposure to a number of envi-ronmental toxins and carcinogens. In phase I the sub-strates are mostly metabolized to highly toxic products,enhancing the significance of the second detoxificationstep.Genetic polymorphisms can cause a malfunction inenzyme production resulting in an insufficient amount ofenzyme or a reduced activity. They have been detectedin a great number of phase I, mainly cytochrome-P450-related enzymes (CYPs), and phase II enzymes asGlutathione-S-transferases (GSTs) and N-acetyltrans-ferases (NATs).The family of the Glutathione-S-transferases operates incellular protection against oxidative stress, pollutans, andtoxic external chemicals like heavy metals.In our laboratory we detect polymorphisms of the mostrelevant metabolizing genes by real time PCR. We aim toindividuate risk factors of dispositions to diseases beforethey appear, but also to enable and sustain a specifictherapy for known diseases or intolerances.In our new purpose we try to answer question of how wecan use the predefined genetic difference in ability ofdetoxification, to explain why two persons, exposed to thesame environmental factors, react in a different manner.Anyway we recommend genetic analysis and invite peo-ple to make use of them to preserve the own health.

CHARACTERIZATION AND SORTING OF FLOWCYTOMETRIC POPULATIONS OCCURRING INHUMAN SEMENMuratori M.1, Marchiani S.1, Tamburrino L.1, Betti L.2,Olivito B.2, Azzari C.2, Forti G.1, Baldi E.1,1Dept. of Experimental and Clinical Biomedical SciencesAndrology Unit, University of Florence, viale Pieraccini6,I-50139 Florence. Italy 2Department of HealthSciences, A. Meyer Children’s Hospital, Florence, Italymonica.muratori@unifi.it

Present tests for the diagnosis of male infertility areunable to reveal all the sperm traits that are needed tosuccessfully reach the oocyte and properly deliver anintact paternal genome. Flow cytometry (FC) couldaccomplish this goal, thanks to its ability to simultaneous-ly measure many parameters at the single cell level.Human semen may contain both somatic and germ cells,including immature elements other than spermatozoa.FC reflects the complexity of human semen in the linearFSC/SSC cytograms, where spermatozoa are containedwithin a flame shaped region (FR). In the latter, by simplystaining nuclei in membrane permeabilized cells, furtherFC subpopulations can be observed: anucleated ele-ments and two populations that differ by the nuclearlabeling with propidium iodide (PI), termed PIbrighter andPIdimmer populations. In this study, we further character-ized these semen FC populations. First, we verifiedwhether FR does include all semen spermatozoa. Weadded fluorescent beads in PI stained sperm and then wecalculated the ratio: nr sperm/nr beads by both lightmicroscope, LM (r1) and FC within the FR (r2). We foundthat in all samples where the FR was clearly depicted, thevalues of r1 and r2 were similar, indicating that FR spermoverlap all semen sperm. Secondly, we demonstratedthat PIdimmer elements are actually spermatozoa by sort-ing PI stained semen samples by FACSAria II andobserving them directly with LM. Finally, we studied thecause of the lower staining in PIdimmer sperm, by hypothe-sizing that such sperm lost DNA fragments, due to aprocess of testis apoptosis interrupted during chromatinmaturation. To investigate the presence of apoptosis andimmaturity, we labeled sperm with FLICA and, in sortedPIdimmer sperm, with aniline blue. We found that PIdimmer

sperm express signs of both immaturity (43.4±19.8%,n=5) and, above all, apoptosis (77.2±9.3%, n=9), sug-gesting that such population could derive by testisabortive apoptosis.

MECHANISMS ORIGINATING HUMAN SPERM DNAFRAGMENTATIONMuratori M.1, Marchiani S.1, Tamburrino L.1, Cambi M.1,Olivito B.2, Azzari C.2, Forti G.1 and Baldi E.11Department of Experimental and Clinical BiomedicalSciences, University of Florence, Florence, Italy.2Department of Health Sciences; University of Florence;Florence, Italymonica.muratori@unifi.it

Sperm DNA fragmentation (sDF) negatively impacts onhuman reproduction. The causes of sDF are not yet fullyclarified, even if several mechanisms have been hypoth-esized. sDF could be due to an apoptotic process, asDNA cleavage is one of the late steps of this type of celldeath. Another proposed mechanism is the failure in there-ligation of those DNA nicks that are produced to favorthe replacement of histones by protamines during spermchromatin maturation. Finally, sDF could result from the

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CD45 SHIFTED EXPRESSION IN AN ATYPICAL CASEOF AGGRESSIVE NK LGL LEUKEMIABencini S1, Bonetti MI1, Mannelli F1, Peruzzi B2, Cutini I1,Gianfaldoni G1, Gelli AMG2, Caporale R2 and Bosi A1.1Hematology Unit, AOU-Careggi; 2General LaboratoryUnit (Microscopy and Clinical Cytometry Unit) AOU-Careggi, Italysara.bencini@virgilio.it

Background: Aggressive NK LGL (large granular lympho-cytes) leukemia (ANKL) is a rare clonal lympho-prolifera-tive disorder which represent less than 0.1% of all lym-phoid neoplasm. ANKL is characterized by a systemicproliferation of NK cells that can involve both peripheralblood (PB) and bone marrow. Typically neoplastic cellsshow a CD45+, CD2-, sCD3-, cytCD3+, CD56+, CD57-,CD16+, phenotype with germ-line TCR genes. The dis-ease course is typically fulminant with a very poor prog-nosis.Aim: The aim of this study is to demonstrate the presenceof a very rare population of NK cells with an atypical phe-notype in the PB of a patient that two months later devel-oped an ANKL.Method: Two different flow cytometry analysis were per-formed in the PB of the same patient, respectively inJanuary and March 2013. The antibodies panel includes:CD2, sCD3, CyCD3, CD5, CD7, CD4, CD8, CD16,CD56, CD57, CyTdT, CD158a and CD158b. Data acqui-sition and analysis were performed using a FACSCANTOII flow cytometer and FACSDIVA software.Results. In January, a neoplastic NK cells population(0.3% of the total cells analyzed) was detected in the PBof the patient showing the following immunophenotype:sCD3-, CD7++, CD16+, CD56++, CD2-, CD5-, CD4-,CD8+dim, CyTdT-, CyCD3-, CD158a+, CD158b+. Thisrare cell population was aberrantly negative for CD45antigen. Two months later, the leukemic cell populationwas increased to 23% of the total PB cells; the phenotypewas concordant with the previously, except for the CD45expression that was shifted to the positivity.Conclusions. New diagnostic strategies need to be inves-tigated in order to improve the outcome of ANKL patient.This ANKL case with an atypical and very rare CD45 neg-ative immunophenotype at presentation can be useful toupdate the diagnosis management of this such aggres-sive disease.

MONITORING OF CHIMERISM ON SORTED PERIPHE-RAL CD34+ CELLS IN PATIENTS WITH ACUTE LEU-KEMIA RECEIVING ALLOGENEIC BONE MARROWTRANSPLANT: A CASE REPORTBonetti M.I1, Peruzzi B.2, Mannelli F.1, Lari A.3, RondelliT.2, Bencini S.1, Cutini I.1, Caporale R.2, Gelli A.M.G.2,Torricelli F.3 and Bosi A.11Unità Funzionale di Ematologia, Università degli Studi,AOU Careggi, Florence, Italy, 2SOD LaboratorioCentrale, Settore Citometria Clinica, AOU Careggi,Florence, Italy, 3SOD Diagnostica Genetica, AOUCareggi, Florence, Italymarida.bonetti@libero.it

Background. An early detection of neoplastic cells afterallogeneic hematopoietic stem cells transplantation

Hematology

attack by reactive oxygen species (ROS), when the latteroverwhelm the sperm antioxidant defenses. In this studywe investigated these putative mechanisms of origin ofsDF, using sperm sorting and tri-parametric analyses bythe FACSAria instrument. The roles of apoptosis andoxidative stress were studied in sperm labeled by DAPI,TUNEL and, respectively, FLICA (a fluorescent inhibitorof caspases, CA) and a fluorescent secondary antibodyrevealing 8-hydroxy,2-deoxyguanosine (8-OHdG), thehallmark of oxidative DNA damage. To understandwhether sperm with DF are immature, DAPI/TUNELlabeled semen samples were sorted to obtain sperm withand without DF, and the two fractions were stained withaniline blue (AB), that labels the excess of histones insperm nuclei. Concerning the apoptotic pathway, wefound that virtually all spermatozoa with DF express alsothe apoptotic enzymes (n=7). Conversely, the co-localiza-tion of 8-OHdG and sDF was found in a very small frac-tion of spermatozoa (3.5±2.0%, n=9). Finally, we foundthat in sperm with and without DF, AB labeling wasrespectively 76.5±18.6 and 37.2± 16.6 %, n=6 (p<0.001).This is the first report that directly investigated the caus-es of sDF. Apoptosis and, at a lesser extent, the impair-ment during chromatin maturation seem to be the mainmechanisms responsible for sDF, whereas a direct role ofROS in inducing sDF before ejaculation appears to bemarginal. However, ROS may indirectly induce sDF bytriggering the apoptotic pathway.

SILVER NANOPARTICLES-INDUCED CHLOROPLASTSDAMAGE IN LEMNA MINOR: AUTOFLUORESCENCESPECTROSCOPY AND IMAGING BASED ONCONFOCAL AND TWO-PHOTON LASER SCANNINGMICROSCOPYRamoino P.1, Bianchini P.2, Gambardella C.3, Diaspro A.2,Falugi C.1, Gatti A. M.4, Tagliafierro G.1, Usai C.51DISTAV, Univ. of Genoa, Italy; 2Ital. Inst. Techn., Genoa,Italy; 3ISMAR, CNR, Genoa, Italy; 4Nanodiagnostic srl,Modena, Italy; 5Inst. of Biophysics, CNR, Genoa, Italyusai@ge.ibf.cnr.it

Silver nanoparticles (AgNPs) are extensively applied asbactericides or fungicides. Moreover AgNPs areemployed in merchandise like household appliances,cleaners, clothing, cutlery, children’s toys, and coatedelectronics. As a result of this extensive application, alarge fraction of the AgNPs commonly end up into aquat-ic ecosystems, where, due to their anti-bacterial proper-ties, probably exert negative effects. In this study the tox-icity of a suspension of AgNPs on the aquatic plant modelLemna minor (duckweed) was investigated. The effectsof different concentrations of AgNPs (0.1-10 µg mL-1) onthe Lemna leaves were evaluated after 7-days exposureby analyzing autofluorescence spectra and optical imag-ing of L. minor via confocal and two-photon laser scan-ning microscopy. Fluorescence spectra were similar bothin control and treated groups, but the fluorescence peak,around 685 nm, was less intense in treated plants.AgNPs-induced inhibition of autofluorescence in Lemnawas dose-dependent. Furthermore, three-dimensional(3D) construction of the entire chloroplast structure bystacks of optical slices, shows that AgNPs-treated andcontrol chloroplasts differ in thylakoid organization.Control chloroplasts display a fluorescence condensationwith brightly appressed areas clearly separated by darkspaces, while thylakoid structure is completely disorgan-ized in 10 µg mL-1 AgNPs -treated chloroplasts. ThusLemna minor is a good biosensor for an accurate evalu-ation of environmental toxicity by metallic nanoparticles.

(HSCT) in adult patients affected by acute leukemia pro-vides the opportunity to manage and control the potentialre-emergence of malignant clone. Several groups havereported as rapidly increasing mixed chimerism correla-tes with higher risk of relapse.Aim. Some predictors of impending relapse are monitoringof Minimal Residual Disease (MRD) by flow cytometry (FC)and PCR-based chimerism on both Bone Marrow (BM) orPeripheral Blood (PB) samples at specific time-points afterHSCT. Restricting chimerism analysis to sorted CD34+ cellscould be an early predictor of relapse after HSCT.Methods. At diagnosis was established for each patientthe most useful leukemia-associated aberrant immuno-phenotype (LAIP) to be investigated at specific time-points during treatment plan to detect LAIP-positive cells.DNA was extracted from PB of donor and recipient beforeHSCT and chimerism assessment was performed onwhole PB and sorted CD34+ cells of recipient after HSCTat specific time points: day 30, 60, 100, 180, month 12and 18 from HSCT. The project is funded by Ministerodella Salute and Regione Toscana (CUPD11J09000190003).Results. The case we report on was enrolled in the proj-ect at April 2012. At day 30 from HSCT (matched unrela-ted donor), the chimerism on whole PB was 100% donorwith MRD-negative; of note, chimerism on sorted CD34+cells was 60% donor. At day 60, FC-MRD converted topositive (0.23% of global cells) whereas chimerism onwhole PB was still 100% donor. At day 100, chimerism onwhole PB decreased and the patient experienced BMrelapse. Chimerism analysis on sorted CD34+ cells allo-wed a very early diagnosis of disease recurrence byhighlighting the presence of the patient’s cells at the firsttime-point with concomitant “negative signals” from FC-MRD and whole PB chimerism.

CD200 EXPRESSION IN MYELOID LEUKEMIC BLASTSBruno G., Colarossi S., Palumbo G., Mangialetto N.,Capalbo S. F.Laboratorio c/o Struttura Complessa di Ematologia,Azienda Ospedaliero-Universitaria di Foggia.peppe.f-12@hotmail.it

INTRODUCTION. CD200 is a trans-membrane glycopro-tein belonging to the type-1 immunoglobulin superfamilyand is expressed on many different cell types including Tand B lymphocytes and dendritic cells. The overexpres-sion of CD200 has been implicated in the pathogenesisof solid tumors and hematological malignancies includinglymphoma, myeloma, chronic lymphocytic leukemia andacute myeloid leukemia (AML).AIM. Assess CD200 antigen expression on AML blastsand correlation of CD200 expression with AML subtype.METHODS. The study included 44 patients, 21 male and23 female, with a median age of 68 years (range 16-94).The FAB classification system divides our cohort ofpatients into six subgroups: 5, 6, 14, 5, 8, 6 subjects wererespectively included in M0, M1, M2, M3, M4, M5 cate-gories. Multiparameter flow cytometry analysis of bonemarrow and peripheral blood was performed using apanel of monoclonal antibodies included CD200, CD13,CD33, CD34, CD14, CD117, HLA-DR, in order to assesantigen expression on AML blasts.RESULTS. Blast cells of 21/44 patients (48%) expressedCD200. The expression of CD200 in the FAB subgroupswas 5/5 in M0 (100%), 5/6 in M1 (83%), 5/14 in M2(36%), 2/5 in M3 (40%), 4/8 in M4 (50%), 0/6 in M5 (0%).CD200 was co-expressed with staminality markers suchas CD34 and HLA-DR. In 19 out of 21 patients CD200

was co-expressed with CD34 (CD34 was no expressed inthe two CD200-positive M3). All CD200-positive samplesexcept one (M3 patient) also resulted HLA-DR-positive.Only three subjects expressed CD200 and CD14 (mono-cytes marker). CD200 was co-expressed with CD13 andCD33 in 18/21 and 15/21 patients respectively. CON-CLUSIONS. CD200 could be considered as a marker ofmyeloid blast cells less differentiated and introduced intothe panel of immunophenotypic characterization of AML.Further studies are need to investigate the prognostic sig-nificance of this antigen.

SPONTANEOUS REGRESSION (SR) OF CHRONICLYMPHOCYTIC LEUKEMIA (CLL) TO MONOCLONALB-CELL LYMPHOCYTOSIS (MBL): REPORT OF TWOCASESD’Arena G.1, D’Auria F.2, Statuto T.2. Guariglia R1,Pietrantuono G.1, Mansueto G.1, Martorelli M.C.1, Villani O.1,Musto P.11Department of Onco-Hematology, 2Laboratory andTranslational Research, IRCCS “Centro di RiferimentoOncologico della Basilicata”, Rionero in Vulture, Italygiovannidarena@libero.it

SR of CLL to MBL is rarely seen: 14 patients have beenreported so far (Nahkla, 2013). We report herein 2 addi-tional patients showing SR to MBL after the initial diagno-sis of early stage CLL. The first patient was a 57-year oldwoman diagnosed with Rai stage 0 CLL in October, 1999.She had a white blood cell count (WBC) of 10x109/L(6x109/L were CD19+CD20+CD23+CD5+ B-lympho-cytes, with kappa light chain restriction). The patient wasthen managed by observation only; she maintained a sta-ble lymphocyte count until December, 2006, when WBCcount and differential were found normal (WBC5.4x109/L). However, a clonal B-cell population wasshowed (2.4x109/L). A normal WBC count was alwaysfound until the last evaluation (August, 2013), despiteflow cytometry monitoring showed the persistence of asmall population of clonal B-cells. The second patientwas a 75-year old woman diagnosed with Rai 0 CLL inJanuary, 2002. She had a WBC count of 10.6x109/L(7,1x109/L were CD19+CD20+CD23+CD5+ B-lympho-cytes, with kappa light chain restriction). The patient wasfollowed-up with routine laboratory and clinical examina-tion; she maintained a stable lymphocyte count untilOctober 2009, when WBC count was found normal(4.3x109/L), with a mild lymphocytosis (45%) at differen-tial count. From May, 2012 the patient maintained a nor-mal WBC count until the last evaluation (August, 2013).Also in this case, periodical immunophenotyping showedthe persistence of a small population of clonal B-cells. Inboth cases FISH analysis (for del13q14, del17p, del11q,trisomy 12) was negative, IgVH mutational status wasmutated and either CD38 and ZAP-70 were negative. Inconclusion, CLL is a neoplastic disorder with significantclinical and biological heterogeneity. The better knowl-edge of the factors able to induce SR or other types ofregressions in this lympho-proliferative disorder, even inpatients with advanced disease, could open the way topossible novel and targeted therapeutic strategies.

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A SHORTER TIME TO THE FIRST TREATMENT(TFT)MAY BE PREDICTED BY THE ABSOLUTE NUMBEROF REGULATORY T-CELLS (TREGS) IN PATIENTSWITH RAI STAGE 0 CHRONIC LYMPHOCYTICLEUKEMIA (CLL)D’Arena G.1, D’Auria .F2, Statuto T.2, Simeon V.3, Musto P.11Department of Onco-Hematology, 2Laboratory of ClinicalResearch and Advanced Diagnostics, and 3Laboratory ofPreclinical and Translational Research, IRCCS ‘‘Centro diRiferimento Oncologico della Basilicata’’ Rionero inVulture, Italygiovannidarena@libero.it

Tregs are increased in CLL and correlates with clinicaland biological features of active/progressive disease. Weevaluated a cohort of 75 consecutive patients (pts) withRai stage 0 CLL, in whom the absolute number of Tregswas determined at the time of diagnosis and correlated tomain clinical and biological features, as well as to theneed of receiving any specific therapy. Overall, 12 pts(16%) did reach a point during the follow-up (mediantime: 16 months) to need therapy. One pt was treatedafter just 2 months from diagnosis because of a suddenand relevant increase of lymphocyte count (from 105,400to 189,200/µL). Three pts doubled lymphocytes after 5, 6,and 7 months, and then treated. The remaining 8 ptsunderwent therapy because of they shifted to a moreadvanced clinical stage, along with a relevant, progres-sive lymphocyte increase and appearance of clinicalsymptoms. None of these events occurred in the cohortof pts who did not receive therapy. Age, gender, and lac-tate dehydrogenase serum levels were not statisticallydifferent between untreated and treated pt groups (p <0.14 and 0.36, respectively). Treated pts, however,showed a higher number of white blood cells and B-celllymphocytes (p < 0.001), lower hemoglobin values (p <0.015) and higher platelet count (p < 0.012). With respectto other biological prognostic factors, a greater number ofcases with unmutated IgVH status (p < 0.025) and carry-ing high risk cytogenetic abnormalities (P < 0.05) wasfound in pts requiring therapy. By contrast, ZAP-70 andCD38 expression was not able to show any statisticallysignificant difference between the two groups.Multivariate analysis confirmed that the absolute numberof Tregs was an independent predictor of TFT in RaiStage 0 CLL pts (p < 0.001). Finally, the best predictivecutoff of Tregs cells was 41/µL. These data show that theabsolute Tregs cell number is able to identify Rai stage 0CLL pts at higher risk of requiring therapy.

VALIDATION PROCESS OF STEM CELLS ENUMERA-TION/QUANTIFICATION: EXPERIENCE OF A SINGLECENTREDattola A., Martino M., Fedele R., Moscato T., Imbalzano L.,Ressa G., Cornelio G., Princi D., Monteleone R., Irrera G.Centro Trapianti Midollo Osseo “Alberto Neri”, AziendaOspedaliera “Bianchi-Melacrino-Morelli”, ReggioCalabria, Italydattola.antonella@libero.it

Aim: In order to achieve JACIE accreditation was mandatoryto validate the process of stem cells quantifica-tion/enumeration (ISHAGE Protocol). The study is retrospec-tive and two years of transplant activity have been analyzed.Method: ISHAGE Protocol (Stem Kit Enumeration–Becton Dickinson) was used; our laboratory participatesto UK NEQAS external quality controls program. In thisstudy were analyzed n. 165 patients undergone autolo-gous transplant

Of each patient are taken into account: peripheral venousblood before harvest, apheresis product (HPC-A), the post-harvest peripheral venous blood and HPC-A infused. Theentire route is on the same day. Of all types of samples areconsidered the following parameters: viable TNC viable %,(7-AAD), viable MNC % (7-AAD), viable CD34 % (7-AAD),enumeration/quantification of CD34+ (absolute value andpercentage), MNC % in the HPC-A.Results: With regard to peripheral venous blood pre-har-vest: TNC is 99%; MNC is 100%; CD34+ is 98.6%; enu-meration/quantification of CD34+ cells is 137 (cells/ul)and 12:31% (total population).With regard to peripheral venous blood post-harvest: theTNC is 98.6,%; MNC is 100%; CD34+ is 98.6%; enumer-ation/quantification of CD34+ cells is 61 (cells/ul) and0.2% (total population).With regard to HPC-A: TNC is 98.2%; MNC is 99.7%;CD34+ is 97.8%, the enumeration/quantification CD34 +is 2134 (cells/ul) and 1.94% (total population).With regard to HPC-A infused: TNC is 66.1%; MNC is88.7%; CD34+ is 68.3%, enumeration/quantification ofCD34 + is 1401 (cells/ul) and 1.52% (total population).The values are expressed in terms of the median.Conclusions: The flow-cytometry performance for the val-idation of the results required at the end of a HPC collec-tion is considered good: there was no “graft failure” andengraftment were in due time.

ROLE OF CIRCULATING ααββ-DOUBLE NEGATIVE TCELLS (DNT) IN LYMPHOMA PATIENTS: PRELIMI-NARY RESULTS OF A PROSPECTIVE STUDYDe Tullio G., Serratì S., Minoia C., Merchionne F.,Loseto G., La Pietra A., Rana A., Iacobazzi A.,Iacopino P., Guarini A.Department of Clinical and Experimental Oncology,Haematology Unit, Advanced Cell Therapy Center,National Cancer Research Centre, Istituto Tumori“Giovanni Paolo II”, BARI, Italyminadetullio@hotmail.com

Many aspects of lymphoma pathophysiology indicatemutual interactions between the host immune systemand lymphoma cells. An unconventional subset ofCD4−CD8− double-negative T cells (DNTs) has beendescribed to specifically contribute to anti-tumor immuni-ty, acting as both regulatory T cells and/or cytotoxic Tcells. No data are available on their role in human anti-lymphoma immunity, their interaction with other immunecells, functional attitude and prognostic significance inlymphomas.The aim of study is to assess the frequency and the func-tional attitude of circulating DNTs in Lymphoma patients(pts) in order to evaluate their predictive role on clinicaloutcome.Peripheral blood of 30 Lymphoma pts and 16 healthydonors were prospectively collected for phenotypic andfunctional characterization of DNTs by staining with:CD3,CD4,CD8,CD56,CD45,TCRαβ,CD45Ra,CD45Ro,CCR7,CD27,CD28,CD30,CD69,GITR,CD95,CD178,CD152,IFN-γ,TNF-α,granzymeB,perforin.For functional stud-ies, DNTs were purified from PBMCs of pts by immunemagnetic selections. Data were acquired using a 8-colourflow cytometer.We observed a significant decrease (p = 0.006) of αβ-DNTs in the PB of pts with untreated lymphoma (20.5 ±4.8 SE,) as compared with healthy controls (31.3 ±3.4),and their number correlated with diseaserelapse/progression. In Hodgkin’s Lymphoma pts the αβ-DNTs frequencies were significantly increased as com-

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pared with other histotypes.Furthermore in ex-vivoexpanded DNTs was observed a characteristic cytokinesprofile with a cytotoxic potential mainly characterized bythe increase of IFN−γ and granzyme B secretion.Our study has demonstrated for the first time that αβ-DNTs may play an important role in both the developmentand the progression of lymphomas. In addition, in ourpreliminary results, it is likely that ex-vivo expanded DNTsexert an anti-tumor activity, thus suggesting their possibleuse as new strategy for adoptive immune-therapy.

PERIPHERAL BLOOD MONOCLONAL T LYMPHOCY-TOSIS AND RISK OF T LYMPHOPROLIFERATIVE DIS-EASE DEVELOPMENTDi Gaetano R., Gasparetto V., Candiotto L., Callegari B.,De Benedetto M.A., Tagariello G.1U.O. Trasfusionale e Immunologia, Centro Regionaleper le Malattie del Sangue, ULSS 8 Veneto, CastelfrancoVenetorosa.digaetano@ulssasolo.ven.it

Background: Occasional evidence of monoclonal gam-mopathy of undetermined significance (MGUS) or mono-clonal B lymphocytosis (MBL) are considered predisposingto the development of multiple myeloma and chronic lym-phocytic leukemia (CLL) respectively and it is recommend-ed to monitor them for possible early diagnosis. In cuta-neous T lymphoma (CTL), such as mycosis fungoides andSezary syndrome, has been demonstrated the presence ofclonal T lymphocytes even in the peripheral blood.Aims: It seems possible that the detection of asympto-matic clonal proliferations lymphocytosis attributable toCTL may represent a predisposing condition to the devel-opment of cutaneous lymphomas.Methods: Every year we analyze by flow cytometry about1700 samples. Patients with peripheral lymphocytosisundergo a standard panel for T, B and NK cells. If clonalB is excluded, we proceed to phenotypic T CD4 subsets(CD7, CD26), analysis of V chains and TCR when neces-sary.Results: From January 2010 to September 2012 weobserved 28 cases of CD3 T> 80% of total lymphocytes(CD3 = 3.000/mm3). Lymphocytosis was variablebetween 3,000 and 10.000/mm3, with an average of5,350. 14 subjects with CD4+CD8- > 75% of T cellsshowed CD7 low/neg, CD26 low/neg- and TCR V sug-gestive of monoclonality, then confirmed by molecularbiology. These subjects were followed up with bloodcount and clinical / dermatological evaluationtwice a year in the event of increased peripheral lympho-cytosis or appearance of symptoms suggestive of lym-phoma. Two patients developed erythematous skinlesions and skin biopsy diagnosed the presence ofSezary syndrome, also confirmed on lymph node andbone marrow biopsies. Both these patients belonged tothe subgroup with CD3 + CD4 + CD7-/ + CD8-CD26-phenotype, typical of Sezary syndrome.Summary / Conclusion: 7.1% (2/28) of patients with mon-oclonal T lymphocytosis have developed a Sezary syn-drome in a median follow-up of 20 months. Based onthese preliminary data the occasional finding of T lym-phocytosis with “atypical” phenotype CD3 + CD4 + CD7-/ + CD26-, may be a useful tool in order to allow earlydiagnosis of CTL in analogy with the strategy currentlyused for patients with MBL. If the hypothesis will be con-firmed in larger cohort this approach could represent anadvantage in terms of early diagnosis and timely treat-ment in this subset of patients.

PROANGIOGENIC EFFECT OF BONE MARROWFIBROBLASTS IN PATIENTS WITH MULTIPLEMYELOMADi Marzo L., Rao L., Ria R., Basile A., Catacchio I.,Solimando A., Vacca A., Frassanito M. A.. Dipartimento di Scienze Biomediche e Oncologia Umana,Clinica Medica “G. Baccelli”, Azienza OspedalieraConsorziale-Universitaria Policlinico di Bari, Italya.frassanito@dimo.uniba.it

Bone marrow (BM) stromal cells favor progression ofmultiple myeloma (MM) but cancer-associated fibroblasts(CAFs) have not been studied yet. CAFs were increasedin patients with active MM (at diagnosis, at relapse) com-pared to those in remission, those with monoclonal gam-mopathy of undetermined significance (MGUS), andthose with deficiency anemia. They displayed an activat-ed phenotype, and produced high levels of TGFβ, IL-6,SDF1α, and IGF1. They showed a heterogeneous phe-notype which entailed their origin from resident fibrob-lasts, and from endothelial cells (ECs) and hematopoiet-ic stem and progenitor cells via endothelial-mesenchymaltransition, and from mesenchymal stem cells via mes-enchymal transition, all induced by CAFs themselves andMM cells. Active MM CAFs induce proliferation and apop-tosis-resistance of MM cells through cytokines and cell-to-cell contact. Studies in syngeneic 5T33MM andxenografted mouse models showed that MM cellsinduced the CAFs expansion which, in turn, favored MMinitiation and progression as well as angiogenesis. In vivoMatrigel plug assays and in vitro chemotaxis showed theability of active MM CAFs (as cells and CM) to attractmouse and human CD31+ ECs, and have a direct angio-genic effect. They support MM tumor growth by promot-ing its neovascularization and contribute to the angio-genic switch and the subsequent angiogenic phase whichparallel transition of MGUS into MM. Moreover inpatients’ and mice BM biopsies nests of CAFs were foundin close contact with MM cells suggesting a vascular pro-tective niche. Targeting CAFs in MM patients may beenvisaged as a therapeutic strategy.

COMPARISON OF DIFFERENT PLATFORMS IN FLOWCYTOMETRY FOR ENUMERATION OF RESIDUALLEUCOCYTES IN LEUCO-DEPLETED BLOOD COM-PONENTSDi Terlizzi S.1, Sizzano F.1, Canonico E.1, Palini A.2, Villa C.11Flow cytometry Resource, Advanced CytometryTechnical Applications Laboratory(FRACTAL) Istituto Scientifico San Raffaele, Milano, Italy.2Flow Cytometry Unit, Nestlè Institute of Health Sciences,Lausanne, Switzerland.diterlizzi.simona@hsr.it

A reliable method to count low levels of white blood cells(WBCs) in platelet concentrates (PCs) and leucocyte-depleted red cell concentrates (RCCs) is essential forQuality Assessment of these leuko-depleted products. Acomparison was performed using two different countingmethods. The first method was based on pre-counted flu-orescent beads (FlowCount® beads) and was performedon two flow cytometer analyzers that introduce sample bya pressure differential. The second was based on a volu-metric device (Partec CyFlow® Cube6) whereby ananalysis and count is performed on a fixed volume (asdefined by the distance between two platinum electrodes)or a desired volume may be freely selected by software.For both methods, PC and RCC samples were labeledwith a mixture containing a membrane permeabilizing

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agent, RNAse and propidium iodide to discriminate thenucleated cells. WBC absolute numbers obtained withthe addition of beads were derived from the ratio of cellsto beads. True volumetric absolute counting (TVAC) ofWBC’s was performed with the addition of 500uL of PC orRCC sample prepared with a mixture provided by Partecto the Cube6 Instrument. A fixed volume of sample(200uL and/or 800uL) was determined automaticallythrough the passage of the sample between two elec-trodes or determined by software. WBC numbers wereobtained directly and reported as WBCs/mL as displayedat the end of acquisition.Prior to experimentation, peripheral blood from a healthydonor was used to set up the appropriate gating strategyon the different instruments.There was a positive correlation in the number of leuco-cyte counts between the compared counting methodsand instrumentation. We also found a good correlation (r> 0.90) between the two counting methods tested on thevolumetric device (electrodes and software control).In conclusion, the Cube6 instrument provides an alterna-tive rapid and reliable method for counting very low num-bers of leucocytes in leuko-depleted products.

A IMMUNOPHENOTYPIC VARIANT HAIRY CELLLEUKEMIA. ROLE OF THE CYTOMETRY Fadda M.R.*^, Prezzemolo T.°^, Lo Presti E.°^, Saieva L.°^,Mitra M.E.*, Siragusa S.*, Dieli F.°^, Caccamo N.°^.*Haematology Unit, University of Palermo, Italy.°Department of Biopatology, University of Palermo, Italy.^Central Laboratory of Advanced Diagnosis andBiomedical Reserch; University of Palermo, Italyraffada@libero.it.

Hairy cell leukemia(HCL) is a mature B-cell non-Hogkinlymphoma characterized by pancytopenia, splenome -galy(96% of cases), lymphadenopathy (35% of cases)and an indolent clinical course. Diagnosis is based onmorphology, bone marrow biopsy(BM) and flow cytome-try (FC). The treatment of choice is purin nucleosid ana-logues which overall responses in >85% of patients.Rituximab has been recently introduced for treatment ofHCL patients prolonging event free survival. We reportthe case of a woman (75 years old) with severe pancy-topenia and splenomegaly. The BM biopsy showed anHCL with an unusual immunophenotypic profile (IIC:CD20+; 4 KB5+; CD3-; CD5-; CD23+; CD25+; CD10+;CD43+/-; BCL6-), therefore FC of peripheral blood wasrequested. Cells were stained with CD3, CD4, CD5, CD8,CD10, CD19, CD20, CD22, CD23, FMC7, CD103,CD11c, CD25, CD38, CD45, κ and λ mAbs and analyzedwith a FACS Canto flow cytometer with Diva software.The analysis showed an infiltrate (5%) of cells with a dimSide Scatter positive for CD103, CD19, CD20, CD22.The 50% of these cells were positive for CD25, CD11c,CD10 and CD23, showing an atypical phenotype. Thepatient started therapy with pentostatin without benefit aspancytopenia and splenomegaly persisted. BM showedthe same of the onset. Therefore therapy with Rituximab,375mg/m2 for 4 cycles, was introduced. Two months afterthe end of therapy, the blood count improved andsplenomegaly was reduced. Therefore, the patient didn’tobtained response to purine analogues therapy, insteadshe obtained response to anti- CD20 therapy. In conclu-sion, FC is a tool with a great diagnostic power and a lowcost and it is an integral component of contemporaryhematopathology practice. We hypothesize that theforms of HCLs, who don’t respond to therapy, need a dif-ferent approach of the clinicians on the use of appropri-

ate therapeutic protocol that can be suggested by theimmunophenotypic analysis.

THE IMMUNOPHENOTYPING CAN BE A PROGNOSTICFACTOR IN ACUTE PROMYELOCYTIC LEUKEMIAFadda M.R.*^, Mitra M.E.*, Di Liberto D.°^, La Manna M.°^,Orlando V.°^, Cannizzo G.^, Siragusa S.*, Dieli F.°^,Caccamo N.°^.*Haematology Unit, University of Palermo, Italy.°Department of Biopathology, University of Palermo, Italy^Central Laboratory of Advanced Diagnosis andBiomedical Reserch; University of Palermo, Italyraffada@libero.it

Acute Promyelocytic Leukemia(APL) is a rare diseasecharacterized by chromosomal translocation t(15;17)leading to fusion gene PML/RARα that responses to mat-uration inducing treatment with all trans-retinoicacid(ATRA). Flow cytometry(FC) mat improve the accura-cy of diagnosis and leads to detection of APL forms withworse prognosis. We present a distinct subtype of APL asdefined by FC analysis, in a young woman with leukocy-tosis, anaemia and thrombocytopenia, cutaneous hemor-rages and low fibrinogen level. The bone marrow smearsrevealed an infiltrate of big granulated blasts. Leukocytecell suspension from peripheral blood(PB) and bone mar-row(BM) were stained with CD2, CD3, CD4, CD7, CD8,CD10, CD11b, CD19, CD20, CD22, CD13, CD14, CD15,CD16, CD33, CD34, CD64, CD56, CD117, HLA-DR,CD45 mAbs and analysed by FC using a FACS Cantoflow cytometer with Diva software. The analysis of PBand BM revealed an infiltrate of immature myeloidcells(84%) CD64+, CD33+, CD13+, CD2+, CD117-. Cellshad an high side scatter like a classic APL form and 16%of these blast cells were CD34+. Patient started AIDA pro-tocol and biomolecular analysis confirmed gene fusionPML/RARα positive after 24 hours. On day 4 patient hadside effects related to ATRA Syndrome and Pseudotumorcerebri(PC). ATRA was discontinuated and Dexameta -sone and Furosemide therapy was promptly started. Onday 6 patient died for intracranial haemorrhage. The blastcells had a pattern of an atypic APL CD34 positive andCD117 negative surface markers. Moreover, blast cellscoexpressed the T lineage CD2 with myeloblastic markertypic of the hypogranular variant form. We ipotize thatimmunophenotipic analysis may be useful for risk stratifi-cation in APL patients, allowing to identify patients withbad prognosis and a major risk of bleeding, ATRA syn-drome or PC. It is necessary to analyse a large numberof cases to include the immunophenotype as individualprognostic factor in APL patients.

CHARACTERIZATION OF BAFF-RECEPTOR EXPRES-SION IN LEUKEMIC LYMPHOID/MYELOID CELL LINESAND IN PEDIATRIC B-CELL ACUTE LYMPHOBLASTICLEUKEMIAFazio G.,1 Cazzaniga V., 1Maglia O.,1 Sala S.,1 Biondi A.,1Gaipa G.1 and Cazzaniga G.11Pediatric Clinic, University of Milan Bicocca, M.Tettamanti Research Center, Monza, Italyg.fazio@hsgerardo.org

B cell-activating factor (BAFF) and its close relative pro-liferation-inducing ligand (APRIL) belong to the tumornecrosis factor (TNF) family, which mediate the behaviorof most B cells and through interactions with their familyreceptors. Among them, only the BAFF receptor (BAFF-R) interacts specifically with BAFF, being the mainresponsible for primary B cell survival, selection and dif-

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ferentiation, in physiology and in disease conditions.We aimed to investigate the potential role of BAFF/BAFF-R axis in Acute Lymphoblastic Leukemia involving B-CellPrecursor (BCP-ALL). With this purpose, we analyzedthe pathway in hematological tumor cell lines well as inprimary BM and PB samples from children affected byBCP-ALL.Preliminary results demonstrated that BAFF-R is highlyexpressed in the B-lymphoid leukemic cell lines, such asREH, TOM1 and NALM-6 (RT-PCR analyses). Its expres-sion is also detectable, although at lower levels, in mixedlymphoid/myeloid phenotype cell lines (such as THP1and RS4;11), in myeloid K562 cells as well as in U937histiocytic lymphoma cell lines. Supported by this data,we further collected BM and/or PB of 24 consecutivediagnostic samplesof Pediatric BCP-ALL. We analyzedBAFF-R expression by flow cytometry, byindirect stainingusing a biotinylated antibody anti-BAFF-R (kindly provid-ed by Prof. Rolink, UniBas, Switzerland) and anti-strepta-vidin-PE as a secondary antibody. In the same samplewe assessed the CD19 (FITC), CD10 (APC) and CD45(PerCP) direct staining to recognize leukemic blast cellsamong the residual of normal cells. For each patient, inaddition to the diagnostic sample, we analyzed at leastone follow up sample (i.e. at day+8, +15 or +33). Wedetected high levels of BAFF-R onCD19+CD10+CD45dim leukemic cells, which persistedduring the follow up treatment. Moreover, we separatedplasma by centrifugation of n=14 patients to analyzeBAFF cytokine levels by ELISA technique, and weobserved higher levels at diagnosis compared to followup samples. In conclusion, we assessed the expression of BAFF-R onleukemic blast cells of BCP-ALL patients. Of note, itsexpression is maintained on residual tumor cells duringthe drug treatment, suggesting the potential targeting ofthis molecule in future advanced treatment approaches.All together, BAFF/BAFF-R axis could have a role in B-ALL, although additional studies are required to compre-hend its role in the pathogenesis of leukemia.

THE INTEGRATION OF: MULTIPARAMETRIC FLOWCYTOMETRY, AUTOMATED CYTOMETRY AND MOR-PHOLOGY IN THE CHARACTERIZATION OF A CASEOF MASTOCYTOSISFumi M., Gaudenzi G., Pancione Y., Sale S., Simeone M.,Tarricone M., Rocco V. Laboratorio di Patologia Clinica – A.O.R.N. “G. Rummo”di Benevento, Italyvincenzorocco1@virgilio.it

Objectives: To explain the rapid variation of the cytogramin a patient in follow-up for MDS after splenectomy.Methods: Woman, 81years, followed by 4 for MDS, in theE.R. because of fatigue, fever and abdominal pain. Onexamination: splenomegaly, for which were performedfurther investigations: CBC made by SIEMENS ADVIA2120i, immunophenotypic characterization with CytomicFC500 B & C, evaluation of peripheral smear.Results and conclusions: CBC, compared with previous,showed a worsening of anemia and thrombocytopenia.Since the patient was known for MDS (RCMD), was notmade morphological evaluation in urgency. Mean while,the CT scan confirmed splenomegaly, with the presenceof two nodules for which was practiced splenectomy. Thefollowing CBC was completely altered compared to theprevious, showing marked increase in LUC andpseudobasofilia. Smear showed cellular elements medi-um/ large, oval / elongated, with thin cytoplasmic ramifi-

cations, round-oval or bilobed nucleus covered with largered-black granules: the morphology suggested the pres-ence of mast cells. Dissolution was immunophenotypewhich showed a population with increased complexity(SSC), positive for CD33, CD117, CD38, CD2, CD25 andnegative for Ag of immaturity / basophils. Positivity forCD2/CD25 likely due to the negative elements for perox-idase and morphologically similar to basophils, suggest-ed the presence of atypical mast cells. All confirmed bybiopsy. In conclusion, although the CBC at entrance inE.R. was compatible with a progression of MDS, the rapidchange of cytographic feature after splenectomy, sug-gested the presence of “atypical” cell populations thatrequired careful characterization. Given the complexity ofthe morphology and the low prevalence of mast cell dis-eases, decisive was contribution of flow cytometry in thecorrect diagnosis of the disease.

INTEGRATING POST INDUCTION WT1 QUANTIFICA-TION AND FLOW CYTOMETRY RESULTS IMPROVESPROGNOSTIC STRATIFICATION IN ACUTE MYELOIDLEUKEMIA (AML)Kunkl A.1, Marani C.2, Clavio M.2, Grasso R2., Colombo N.2,Guolo F.2, Ballerini F.2, Giannoni L.2, Ghiggi C.2, Fugazza G.2,Ravetti J.L.1, Gobbi M.2 and Miglino M.21Diagnostica Citofluorimetrica, Anatomia Patologica,2Dipartimento di Emato-oncologia, IRCCS AOU S.Martino-IST, Genova, Italyannalisa.kunkl@hsanmartino.it

Detection of minimal residual disease (MRD) has a rele-vant prognostic value in AML. In the present retrospectivestudy we evaluated fifty uniformly treated adult non M3AML patients with respect to pretreatment risk factors andpost induction MRD analysis. Forty-two patients (84%)achieving CR after first induction regimen were evaluatedfor reduction of WT1 gene expression (∆WT1=logWT1 atdiagnosis –log WT1 post induction); 34 patients for resid-ual leukemic cells by flow cytometry (flow-MRD) (≥2,5 x10-4 residual leukemic cells defined positive samples).Twenty-three patients (68%) were flow-MRDpos, 11 (32%)were flow-MRDneg. The post induction residual clonalpopulation detected by flow had a strong prognosticvalue: 3-year DFS was 79,5% and 27,3% in flow-MRDneg and in flow-MRDpos patients respectively (p=0.032). None of the patients older than 60 years of agereached a negative flow-MRD, while among 14 high riskprofile patients only one was flow-MRDneg but relapsedafter BMT. In the 23 flow-MRDpos patients, DFS was notaffected by the amount of residual neoplastic population,above or below 1% (p=0.41). Flow-MRDpos patients werestratified according to kinetics of WT1 reduction. Patients(13) with a ∆WT1≥1.5 log had a better outcome com-pared to those (10) with ∆WT1<1.5 log (3-year-DFS was46.2% vs 0%, p<0,001). High risk profile patients at diag-nosis and patients not achieving ∆WT1≥1.5 log post-induction form unique poor risk subgroups of patients thatshould receive alternative treatments including BMT.

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MULTI-LINEAGE DYSPLASIA AS ASSESSED BYIMMUNO-PHENOTYPE HAS NO IMPACT ON CLINI-CAL-BIOLOGIC FEATURES AND OUTCOME OFACUTE MYELOID LEUKEMIA WITH MUTATEDNUCLEOPHOSMIN (NPM1)Mannelli F., Bencini S., Gianfaldoni G., Ponziani V., Cutini I.,Scappini B., Pancani F., Biagiotti C., Bonetti M. I.,Longo G., Bosi A.Hematology Dept, University of Florence, Florence, Italy francesco.mannelli@unifi.it

Background. Acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD) is a separate subset in WHOclassification. Morphologic study of residual hemopoiesisis the standard criteria for defining MLD. Its prognosticvalue is still under debate: i) morphology is operator-dependant; ii) MLD-related poor prognosis is supposed torely on progression from a myelodysplastic syndrome(MDS) but MLD might merely result from maturation byAML clone. A major controversy concerns MLD value inNPM1-mutated (NPM1+) AML, since NPM1+ status cor-relates with good prognosis. Aims. To study MLD inNPM1+ AML by an alternative technique: flow cytometry(FC) is emerging for studying dysplasia. Its application toAML allows: i) to study large cellsí amount; ii) to quantify,refer to controls and standardize FC parameters.Methods. Patients: 70 NPM1+ pts were studied. Flowcytometry: FACSCanto II (BD) and Infinicyt (Cytognos)were used for acquisition and analysis. MLD wasappraised by an immuno-phenotypic score (IPS) includ-ing 18 parameters (14 for granulocytic and 4 for erythroidlines). Results. Median age was 57 (24-70); WBC countwas 48.0 x 109/L (1.2-260.0). Karyotype was normal in62 (88.6%) pts. FLT3-ITD occurred in 27 pts (38.6%). IPSwas calculated in all 70 pts; median IPS was 6.25 (0.5-18.5). Pts were grouped according to IPS higher (IPS+)or lower-equal (IPS-) than the median: age, WBC,platelets, morphologic MLD were not different. IPS+group had lower FLT3-ITD incidence (25.7% vs 51.4%;p=0.048); interestingly, Falini et al (Blood 2010) reportedanalogue results by morphology. CR rate was not differ-ent in IPS- (82.9%) and IPS+ (74.3%; p=0.56) pts. IPSdid not affect disease-free and overall survival.Conclusions. This study provides evidence that MLD, asassessed by FC, does not influence clinical features andoutcome of NPM1+ AML. These findings support NPM1+AML to be considered as a separate entity and its prog-nostic assessment should not be based on MLD.

POST-INDUCTION MRD EVALUATION: A COMPARI-SON OF MULTIPARAMETER FLOW CYTOMETRYAND WT1 RT-PCR TECHNIQUES IN 51 ACUTEMYELOID LEUKEMIA (AML) PATIENTSNuccorini R1, Pascale S1, Coluzzi S1, Matturro A1,Pizzuti M1, Olivieri A2 and Attolico I11U.O. Ematologia AOR San Carlo di Potenza; 2Facoltà diMedicina e Chirurgia Dipartimento Scienze Cliniche eMolecolari, Clinica Ematologica Osp. Regionale Torrette,Italy roberta_n3@yahoo.it

We evaluated post-induction bone marrow mimimalresidual disease (MRD) in 51 AML adults, with medianage of 57 years (range: 22-76). MRD was measured bothby multiparameter flow cytometry (MPFC) and by WT1expression, by RT-PCR. Cytometry tests were performedin 43pts, cytogenetics status was known in 48 pts; NPMand FLT3 status were known in 50pts, defining the molec-ular-cytogenetic risk in 43pts: 18 favorable, 19 intermedi-

ate1, 6 unfavorable. With a median follow-up of 12months (range 2-49), CR rate after induction therapy was84% and Relapse Free Survival (RFS) was 82%; accord-ing to cytogenetics and molecular markers, CR rate was100% in favorable cytogenetic, 86% in NPM+FLT3-, 69%in double negative, 60% in FLT3+, 40% in unfavorablecytogenetics. WT1 was +ve in 36/48 pts (75%) at diagno-sis (median 1155; 51.9-9176) and all pts showed >20%blasts by MPFC. Post induction WT1 was +ve in 4/39(10%) (median 19.6; 0,6-13463.3) and by MPFC MRDwas +ve in 10/36 (28%) pts.MRD evaluated by MPFCand WT1 was concordant in 27/33 (82%) pts after induc-tion. We analyzed the predictive value of post inductionMRD (evaluated by MPFC and WT1) in terms of RFS andOS. In pts with†WT1 -ve (35/39: 90%) OS was 69%(median 20 months; 2-58); in 4/39 (25%) with†WT1 +veOS was 25% (median 7 months; 5-34); MPFC was -ve in26/36 (72%): in these pts OS was 77% (median28months; 2-58); MPFC was +ve in 10/36 (28%): OS was30% (median7 months; 3-34). We observed 9 relapses.Among pts with WT1 -ve RFS was 86% (median22months; 1-57), while in WT1 +ve RFS was 50% (medi-an19 months; 5-33). Among pts with MPFC -ve RFS was96% (median 27 months; 2-48), while in MPFC +ve RFSwas 50% (median 4 months; 1-33). In conclusion, ourdata suggest that WT1 and MPFC are complementarytools for MRD evaluation. Both the techniques were con-cordant in 82% of cases, but MPFC predicts betterthanWT1 the outcome (in terms of OS and RFS respec-tively). These data should be confirmed in a prospectivestudy.

EVALUATION OF BONE MARROW DYSPLASIA BYFLOW CYTOMETRIC ANALYSIS WITH ACCEPTABLEINTER-OPERATOR VARIABILITY: A PROSPECTIVEMULTICENTRIC STUDYPicone C.,1 Tenore A.,1 Geuna M.,2 Girotto M.,3 Stacchini A.,4Rigolin G.M.,5 Ciriello M.,6 Pautasso M.,7 Prato G.,8Rossetto P.,3 D’Auria F.,9 Consensi E.,1 Vanelli L.,1

Del Vecchio L.,10 and Della Porta M.G..11Dipartimento di Ematologia Oncologia, IRCCS PoliclinicoSan Matteo & Università di Pavia; 2Laboratorio diImmunopatologia-Anatomia Patologica, AO OrdineMauriziano, Torino; 3Medicina trasfusionale ed Ematologia,Ospedale di Ivrea; 4Unità di Citofluorimetria, Dipatrimento diPatologia, Azienda Sanitaria Città della Salute e dellaScienza, Torino; 5Ematologia, Dipartimento di ScienzeMediche Arcispedale S. Anna & Università di Ferrara;6Laboratorio di Citofluorimetia, Azienda Ospedaliera SSAntonio e Biagio e C. Arrigo, Alessandria; 7Laboratorio diCitofluorimetria, S. Luigi Gonzaga, Orbassano; 8AnatomiaPatologica-Citofluorimetria, Ospedale S. Andrea, Vercelli;9Ematologia, IRCCS Centro di Riferimento OncologicoBasilicata, Rionero in Vulture; 10Centro di Citometria Clinica eSperimentale, CEINGE & Università Federico II, Napoli, Italyc.picone@smatteo.pv.it

The assessment of marrow dysplasia is the mainstay forthe diagnosis of myelodysplastic syndromes (MDS), andrepresents the basis of the WHO classification of thesedisorders. We conducted a prospective multicentric studyto evaluate the clinical utility of immunophenotyping in thediagnostic and prognostic work up of MDS patients.We first studied a ëílearningíí cohort of 191 patientswhose analysis was aimed at defining the set of cytomet-ric variables with a different expression between MDSand controls affected with non-clonal cytopenia. Analysisof myeloid cells showed increased prevalence of abnor-malities on CD16/CD13 and a CD16/CD11b maturation

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pattern (P<.001), reduced granulocytic SSC (granulo-cytes to lymphocytes SSC peak channel ratio, p<.001)and lower proportions of CD10+ granulocytes (P <.001)in MDS. Analysis of CD34+ cell compartment showedincreased CD45+CD34+ myeloblasts (P<.001), reducedCD34+ B-cell precursors (P<.001), increased expressionof lineage infidelity markers (CD5, CD7, CD19, or CD56,P<.001), and abnormal CD45 expression (lymphocytes tomyeloblasts CD45 ratio, P<.001). Analysis of erythroidcells showed decreased levels of CD71 (P<.001) andincreased expression on cytosolic ferritin (P<.001) onnucleated red cells.The weight in the recognition of bone marrow dysplasiaof each parameter that matched criteria of adequateinter-operator reproducibility (i.e., coefficient of variation<20%) was tested by a multivariable general logisticregression model. A flow cytometric score (FCM-score)for the diagnosis of MDS was defined based on regres-sion coefficients from that model. A score value of 1 wasassigned to CD34+ myeloblasts >1.5%, B cell progeni-tors <4.8% in all CD34+ cells, lymphocytes tomyeloblasts CD45 ratio <4.1 or >7.8, granulocytes to lym-phocytes SCC ratio <6.3, CD10+ granulocytes <28%,CD71 MFI expression on erythroblasts <164, while ascore value of 2 was assigned to CD34+ myeloblasts>3.5%. A diagnosis of MDS was formulated in presenceof a FCM-score value >2We assessed the diagnostic power of FCM-score in theëílearningíí cohort. Overall 115/128 MDS patientsreceived a correct diagnosis (sensitivity 90%). Five false-positive cases were noticed among 63 controls (specifici-ty 92%). In MDS patients, a high score value (>5) wasfound to be associated with the presence of multilineagedysplasia (P=.001), excess blast (P<.001), transfusion-dependency (P=.03) and poor risk-cytogenetic (P=.04),resulting in a higher IPSS risk (P=<.001). An independentvalidation of the obtained FCM-score was carried out ona series of 255 patients (ëívalidation cohortíí). Theobtained sensitivity and specificity were 89% and 91%,respectively. Finally we evaluated the prognostic effect of the FCM-score in 213 patients with a conclusive diagnosis of MDSand complete survival information. In multivariable analy-sis together with demographic factors and IPSS risk, theFCM-score significantly affected the probability of overalland leukemia-free survival (HR 1.91 and HR 2.69,P<.001).These results indicate that immunophenotyping based onparameters with acceptable inter-observer reproducibilitymay help to establish the diagnosis of MDS. Moreover,the evaluation of marrow dysplasia by flow cytometrymay have important prognostic implications

B MEMORY COMPARTMENT AND CD38+ PLASMACELLS IN MULTIPLE MYELOMA PATIENTS Pojero F., Mazzarese M., Salomone R., and Gervasi F. U.O. Laboratorio Specialistico Oncologia Ematologia eColture cellulari per uso clinico, ARNAS Civico, Palermo, Italyfrancesco.gervasi@ospedalecivicopa.org

Introduction: Multiple Myeloma (MM) is characterized bymonoclonal plasma cells accumulation in bone marrow.Recently cancer stem cells (CSC) are attracting attention.In MM peripheral blood (PB) and bone marrow (BM) sam-ples, CD138- B cells sharing same molecular alterationswith MM plasma cells were identified. These cells showproperties of stem cells (side population) and memory Bcells. (Matsui et al. Cancer Res 2008). Given that, westudied B memory compartment.

Material and Methods: Thirty-five MM BM and PB sam-ples were analyzed using the following panels: 1) CD24FITC/ IgD PE / CD45 PercP-Cy5.5/ CD38+CD138APC/CD20 APC-H7/CD19 Pacific Blue; 2) CD27 FITC/CD56 PE/ CD45 PercP-Cy5.5/ CD38+CD138 APC/CD20APC-H7/CD19 Pacific Blue. Almost 1000 on CD19 gateand CD38+138+ were acquired. Acquisition and dataanalysis were performed with CyAN ADPTM and Kaluza1.1 (Beckman Coulter, Miami, FL, USA) respectively.WBC count on PB and BM was performed with DXH800(Beckman Coulter, Miami, FL, USA).Results: Naïve B cells are the most representative popu-lation in PB (M= 57.5%) and BM (M=51.35%). Late mem-ory cells are less represented in PB (M=21.93%) than inBM (M=27.67%). In both PB and BM, according to theBm1-Bm5 classification, B late memory cells are distrib-uted between the gates identifying Bm5 early (BM=7.5%;PB=7%) and Bm5 late (BM=24.2%; PB=24.9%) cells;naïve B cells are included in Bm1 (BM=30.4%;PB=32.6%) and Bm2 (BM=20.23%; PB=24.9%) gates,on the CD38-CD138/IgD dot plot.Conclusions: This study has provided an elegant startingpoint for analysis of CD19+ B lymphocytes antigen-dependent maturation in MM BM and PB, and relation-ship with pathological plasma cells.

USE OF CELL POSITIONAL DATA AND MULTIPARA-METER FLOW CYTOMETRY TO IDENTIFY B CELLCHRONIC LYMPHOCYTIC LEUKAEMIAPojero F., Salomone R., Mazzarese M., and Gervasi F.U.O. Laboratorio Specialistico Oncologia Ematologia eColture cellulari per uso clinico, ARNAS Civico, Palermo, Italyfrancesco.gervasi@ospedalecivicopa.org

Objectives: B cell chronic lymphocytic leukaemia (B-CLL)is a clinical-biological heterogeneous disease. Coulterautomated laboratory analyzers differentiate WBC basedby measures of their volume, conductivity and scatter.We investigate the predictive value of index measures,known as cell population data, compared to morphologicand flow cytometric analysis for detection of chronic lym-phocytic leukemia (CLL).Methods: Morphology, blood cell counts, flow cytometryand cytogenetics analysis were performed in samplesfrom 35 patients with B-CLL and 84 healthy using theBeckman Coulter LH780 and DXH800 and the cytometerCyan ADP TM †(Beckman Coulter, Miami, FL, USA).Volume, Conductivity, Scatter, and antigen distributionpatterns of lymphocytes compared to cytogenetics wereevaluated as predictors of diseaseResults: B-CLL immunophenotype patterns showedCD5+CD23+ homogeneous /spread and CD5+CD23+spread/spread and mean of lymphocytes volume showedmost relevant difference in comparison to healthy sam-ples (p = 0,0001), the area under the ROC curve (AUC)=0,897. No difference appeared in conductivity (mean±SD) and in light scatter (mean±SD) values. Dividingpatients into cytogenetic subgroups according to inter-phase fluorescent in situ hybridization (i-FISH) data, a dif-ference emerged in mean of lymphocyte volume amongpatients carrying del13 and all the others cytogeneticgroups: the del13 group showed a lower mean of lympho-cyte volume in comparison with other genetic group.Conclusions: cell population data and immunophenotypepattern could be routinely used to screen for CLL and tointegrate cytogenetic data.

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EPIDEMIOLOGY OF MATURE B CELL NEOPLASMSIN THE TERNI AREA FROM 2010 TO 2012 USINGFLOW CITOMETRYRicci M., Adami C., Agabiti S., Palmieri G., Scaccetti A. eMaterazzi M.Immunohematology and Blood Trasfusion Unit, FlowCytometry Laboratori S.Maria Terni Hospital, Italymonia_ricci@virgilio.it

We performed a preliminary study to asses the incidenceof mature B cell neoplasms CD5+ and CD5- in the Terniarea in order to later evaluate the impact of environmen-tal contaminants on the incidence and prevalence of suchtumors. We used FACS Canto II BD and the followingantibody panels in Peripheral Blood (PB) samples: for theroutine panel CD3 FITC, CD16PE, CD45PerCP-Cy5,CD4PE-Cy7, CD19APC, CD8APC-Cy7; for the mature Bcell neoplasm panel CD5FITC, CD10PE, CD19PerCP-Cy5, FMC7 FITC,CD23PE, CD38 FITC, CD56PE,CD20APC-Cy7, CD22APC, CD25PE-Cy7, CD11cAPC.Within the population studied, the mature B cell neo-plasms are by far the most frequent findings. This is inagreement with WHO classification of haematopoieticand lymphoid tissues. We diagnosed 160 new cases ofmature B cell neoplasms during the 3 years studied(2010-2012) with an incidence 14, 16 and 17 mature Bcell CD5+ neoplasms /100,000 inhabitants in the year2010, 2011, 2012 respectively. In particular CLL wentfrom 7.7 in 2010 to 10 cases/100,000 inhabitants in 2011with a change in man vs woman ratio (2:1 in 2010 to 1:1in 2011) and a peak of incidence in the population of > 70years old in the studied period. The most frequent pheno-types of CLL were CD20+dim CD22+ CD23+ and Ig k orlambda light chain dim. Mature B cell neoplasms CD5-instead decreased from 7.2-7.2-6.1/100,000 inhabitantsin 2010, 2011and 2012 respectively. The most frequentCD5- B cell neoplastic phenotype were FMC7+, CD20+,CD22+ and CD20+,CD22+. In addition, we found 20 newcases of atypic CLL as defined by the WHO classificationfrom 2010 to 2012.

PROGNOSTIC IMPACT OF MINIMAL RESIDUAL DIS-EASE ASSESSED BY MULTIPARAMETER FLOWCYTOMETRY IN CHILDHOOD ACUTE MYELOIDLEUKEMIARizzati F.,1 Buldini B.,1 Rondelli R.,2 Masetti R.,Francescato S.,1 Zangrando A.,1 Disarò S.,1 MichielottoB.,1 Pession A.,2 Locatelli F.,3,4 Basso G.11Oncoematologia Pediatrica, Dipartimento di Salute dellaDonna e del Bambino, Università di Padova, Italia;2Clinica Pediatrica, Università di Bologna, Ospedale “S.Orsola”, Bologna, Italia; 3Oncoematologia Pediatrica,IRCCS Ospedale Pediatrico Bambino Gesù, Roma,Italia; 4Università di Pavia, Italiafridarizzati@yahoo.it

Background: Achievement of morphologic completeremission (CR) is a prerequisite for cure in acute myeloidleukemia (AML), but patients in CR may still have vari-able levels of occult leukemia, defined as minimal resid-ual disease (MRD). More sensitive methods are requiredto allow detection of leukemic cells: multiparameter flow-cytometry (MFC) is based on the research of aberrantexpression of surface antigens on the malignant cells, toidentify residual cells not detectable by morphologicassessment.Aim: Aim of this study is to evaluate the prognostic impactof MFC-MRD in children with AML.Materials and Methods: Patients younger than 18 years

affected by de novo AML (diagnosis May 2003- May2011), enrolled in AIEOP-LAM 2002 protocol, were eligi-ble for this study.Bone marrow aspirates from 142 patients were collectedat diagnosis, at the end of induction 1 (ICE1) and part atthe end of induction 2 (ICE2). MRD was assessed during follow-up, by five-color MFC,through the detection of patient-specific leukaemia asso-ciated aberrant phenotypes (LAIPs), defined for eachpatient at diagnosis. Results: Among the 142 patients recruited to this study, atthe end of ICE1 35,9% had a level of MRDe”1%, 11,3%with MRD 0.1-1% and 48,6% with MRD<0,1%; 6 caseswere not evaluable. We assessed the correlationbetween levels of MFC-MRD and disease-free-survival(DFS), by means of the Kaplan-Meier method. Presenceof MRD e” 0,1%, at the end of induction 1, was associat-ed with a DFS of 35,29 % + 7,23 % at 6 years, whereasMRD < 0,1% was associated with a DFS of 73,16+ 5,62%at 6 years (p < 0,01). Similar DFS data were obtained atthe end of ICE2 (p < 0,01). Conclusions: Our study revealed a significant correlationbetween MFC-MRD at the end of induction 1, and thepatient’s outcome: MRD≥ 0,1% appeared to be predictiveof poor outcome. Our data suggest that levels of MFC-MRD at the end of ICE1 provide important prognosticinformation that may be used to a better stratification andto guide therapy of pediatric patients with AML.

EXPANSION OF CIRCULATING NATURAL KILLERPOPULATION IN MULTIPLE MYELOMA PATIENTSAFTER LONG-LASTING THERAPY WITH LOW DOSELENALIDOMIDERocco M., Villani G., Rosamilio R., Giudice V., Pezzullo L.,Fontana R., Serio B., Annunziata S., Ferrara I., Selleri C., Hematology and Transplant Center, Department of Medicineand Surgery, University of Salerno, Salerno, Italymonia.rocco@sangiovannieruggi.it

Effects of lenalidomide (LD-R) in Multiple Myeloma (MM)patients are to its direct anti-proliferative and anti-angio-genic properties responsible for cell cycle arrest of myelo-ma cells, and downregulation of IL-6 and VEGF levels inthe tumor microenvironment, respectively. In addition LD-Rmay indirectly increase immunotherapy responses in MMpatients, stimulated T-cell proliferation and enhancing IL-2,IFγ and TNFα secretion by Th1 cells, as well as downreg-ulating regulatory T cells and upregulating cytotoxic effectsof natural killer cells (NKc). We evaluated the numbers ofcirculating NKc in 15 MM (12 males and 3 females) with amedian age of 75 years, during treatment with continuouslow dose of LD-R. Seven of these 15 MM patients werenewly diagnosed MM patients receiving continuous alter-nate-day LD-R (10mg) in combination with low dosesteroids (15mg), the remaining were MM patients receivingcontinuous alternate-day LD-R maintenance therapy afterautologous stem cell transplantation. Flow cytometryanalysis was performed using the Cytomics FC500(Coulter Miami, FL) to determine the expression of T-cellantigens, NKc antigens. As for as T-cell is concerned, wereported the expression of CD3+CD4+ cells for T-helper,CD3+CD8+ cells for T-suppressor, while for the NKc werecorded the expression of CD3+CD56+ cells and CD3-CD56+ for activated NKc. All antibodies were obtainedfrom Beckman Coulter. Our preliminary data show that pro-longed low dose LD-R treatment in MM patients increasescirculating NKc further supporting that this drug may medi-ate its anti-MM effect, at least in part, by modulating NKcnumber and function.

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FINE TUNING OF SURFACE CRLF2 EXPRESSIONAND ITS ASSOCIATED SIGNALLING PROFILE INCHILDHOOD B CELL PRECURSOR ACUTE LYM-PHOBLASTIC LEUKEMIASarno J.,1 Bugarin C.,1 Palmi C.,1 Savino A.,1 Te Kronnie G.,2Biondi A.,1 Basso G.,2 Cazzaniga G.,1 and Gaipa G.11Pediatric Clinic, University of Milan Bicocca,M.Tettamanti Research Center, Monza, Italy - 2Lab.Hemato-Oncology, University of Padua, Padua, Italy j.sarno@campus.unimib.it

Children with B cell precursor acute lymphoblasticleukemia (BCP-ALL) bearing rearranged cytokine recep-tor-like factor 2 (CRLF2) may show high rate of relapse.However, CRLF2 rearrangement is associated with itssurface over expression and aberrant pSTAT5 and pS6signalling pathways. Assessment of phenotypic CRLF2expression and its associated signalling profile may resultin a better understanding of the biochemical mechanismunderlying such aberrancies. A total of 86 bone marrowsamples from children with BCP-ALL were analyzed forCRLF2 expression by flow cytometry (FCM). CRLF2 tran-script levels, as well as CRLF2 aberrations (i.e. P2RY8-CRLF2), were analyzed by RQ-PCR. TSLP-inducedpSTAT5, pS6, p4EBP1 and pAKT473 were evaluated inleukemia blasts by phosphoflow cytometry. Parallel PCRand FCM measurement of CRLF2 expression was per-formed: CRLF2+ samples (6/86) by FCM were RQ-PCRoverexpressed; similarly all CRLF2- samples (77/86) byFCM were concordantly negative by RQ-PCR. An aber-rant TSLP-induced pSTAT5 response was observed in allFCM-CRLF2+ samples tested, and it was significantly dif-ferent compared to CRLF2 negative cases. Interestingly,we detected a subgroup of CRLF2- patients showing aweak fluorescence shift with an intermediate pSTAT5response significantly different compared to both theremaining CRLF2 truly negative patients and CRLF2 pos-itive patients. Furthermore we found a TSLP-inducedpS6, p4EBP1 and pAKT473 response significantly higherin CRLF2+ patients compared to negative. In conclusion,FCM and RQ-PCR provided high concordance in theidentification of patients with CRLF2 over expression.CRLF2+ patients show aberrant pSTAT5 response asso-ciated with significant enhanced pS6, p4EBP1 epAKT473. Interestingly, we identified a subgroup ofpatients CRLF2- by standard cytometric criteria (i.e<10%), showing low CRLF2 expression detectable byassessing accurate fluorescence shift and significantlyhigher pSTAT5 response compared to truly negativecases. This findings may open new opportunities in thescreening of patients that could benefit of signal trans-duction inhibitors treatments.

FLOW CYTOMETRY ANALYSIS OF BRONCHOALVE-OLAR LAVAGE FLUIDS: EVALUATION OF SCREEN-ING PARAMETERS SUGGESTING THE SUSPECT OFPULMONARY B-CELL NON HODGKIN LYMPHOMA Scarpati B.,1 Cesana C.,1 Scampini L.,1 Zilioli VR.,2Rusconi C. ,2 Klersy C.,3 Barba C.,1 Ferri U.,1 Liga G.,1Chiericozzi M.,4 Torre M.,5 Morra E.,2 Vanzulli A.,6 Rossini S.11Laboratorio di Citometria, Dipartimento Medicina diLaboratorio, 2Divisione di Ematologia, OspedaleNiguarda Ca’ Granda, Milano; 3Servizio di Biometria eStatistica, Fondazione IRCCS Policlinico San Matteo,Pavia; 4Divisione di Pneumologia, 5Chirurgia Toracica e6Divisione di Radiologia, Ospedale Niguarda Ca’ Granda,Milano, Italybarbara.scarpati@ospedaleniguarda.it

To find flow cytometry (FC) screening data on bronchoalve-olar lavage fluid (BALF) indicating possible lung B-cell nonHodgkin lymphoma (NHL)(B-NHL), we retrospectivelyselected BALF with pulmonary B-NHL diagnosis at with-drawal (cases), and at least 6 controls per case, matched forcomputed tomography. FC screening included total cellular-ity and CD3-CD4-CD8-CD16/56-CD19 panel, measure-ments containing 50,000 cell events in the list mode storage.Data detected in cases were compared with the median cor-responding values of data detected in controls (Wilcoxontest for paired comparisons).After a median of 18 months, 6 cases (2 mantle cell NHL,1 Burkitt-like NHL, 1 B-cell chronic lymphocytic leukemia, 1diffuse large B-NHL, 1 follicular NHL) were identified (2,ground glass pulmonary infiltrates; 2, enlarged mediastinallymph nodes plus parenchimal densities; 1, nodules; 1,parenchymal density plus nodules). After a median of 21months, 56 (median per case: 10) matched controls (41,infection; 4, sarcoidosis; 3, solid tumor; 8, other) were iden-tified. As compared to controls, cases showed significantlyhigher median lymphocyte (Ly) event number {27,869[interquartile range (IQR) 22,234-40,055] vs 12,103 (IQR8,671-12,924)}, median % of Ly on total leucocytes [65%(IQR 62%-69%) vs 26% (IQR 19%-34%)], median B Lyevent number [997 (IQR 224-2,814) vs 110 (IQR 49-160)],median % of B Ly on total leucocytes [2.9% (IQR 0.7%-4.7%) vs 0.2% (IQR 0.1%-0.3%)], and median T Ly eventnumber [26,657 (IQR 21,699-36,588) vs 11,530 (IQR7,250-11,964)](each P, <.05). Total cellularity (319,900/mLvs 232,053/mL), leucocyte (49,134 vs 49,180) and NK Ly(374 vs 270) events, % of T (58% vs 25%) and NK (0.9%vs 0.7%) Ly on total leucocytes, % of T (92% vs 93%), B(4.6% vs 1%), NK (1% vs 2.3%) Ly on total Ly did not dif-fer significantly between cases and controls.B Ly as event number and as % of leukocytes in BALFseem indicative of performing deepened B-cell phenoty-ing to look for lung B-NHL.

FLOW CYTOMETRY SCREENING OF LYMPHOCYTESUBPOPULATIONS IN BRONCHOALVEOLAR LAVAGEFLUIDS: EVALUATION OF PARAMETERS INDICATIVEOF IN-DEPTH IMMUNOPHENOTYPE ANALYSIS IN THESUSPECT OF PULMONARY T-CELL NON HODGKINLYMPHOMAScarpati B.,1 Cesana C.,1 Scampini L.,1 Zilioli VR.,2Rusconi C. ,2 Klersy C.,3 Barba C.,1 Ferri U.,1 Liga G.,1Chiericozzi M.,4 Torre M.,5 Morra E.,2 Vanzulli A.,6 Rossini S.11Laboratorio di Citometria, Dipartimento Medicina diLaboratorio, 2Divisione di Ematologia, OspedaleNiguarda Caí Granda, Milano; 3Servizio di Biometria eStatistica, Fondazione IRCCS Policlinico San Matteo,Pavia; 4Divisione di Pneumologia, 5Chirurgia Toracica e6Divisione di Radiologia, Ospedale Niguarda Caí Granda,Milano, Italybarbara.scarpati@ospedaleniguarda.it

Flow cytometry (FC) screening data on bronchoalveolarlavage fluid (BALF) suggesting in-depth phenotype lead-ing to prompt lung T-cell non Hodgkin lymphoma(NHL)(T-NHL) diagnosis are undetermined.Among BALF with available follow-up, we selected caseswith pulmonary T-NHL and at least 6 controls per casewith definite non hematologic disease, matched for com-puted tomography. FC screening included total cellularityand CD3-CD4-CD8-CD16/56-CD19 panel, measure-ments containing 50,000 cell events in the list mode stor-age. FC data detected in cases were compared with themedian corresponding values of data detected in controls(Wilcoxon test for paired comparisons).

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After a median of 5 months, 6 cases (each, peripheral T-NHL, n.o.s.) were identified (1, enlarged mediastinallymph nodes; 2, parenchimal densities; 2, ground glasspulmonary infiltrates; 1, parenchimal densities plusground glass infiltrates). After a median of 17 months, 62(median per case: 11) matched controls (40, infection; 6,sarcoidosis; 5, drug-induced pneumopathy; 4, bronchioli-tis; 2, graft rejection; 1, thromboembolism; 1, lung cancer)were identified. As compared to controls, cases showedsignificantly higher median lymphocyte (Ly) event num-ber {18,761 [interquartile range (IQR) 18,611-19,392] vs6,545 (IQR 5,509-10,581)}, median % of Ly on leuco-cytes [41% (IQR 35%-58%) vs 15% (IQR 10%-23%)],median T Ly events [18,126 (IQR 17,756-18,537) vs6,162 (IQR 5,099-10,112)], and median % of T Ly on leu-cocytes [41% (IQR 35%-57%) vs 12.5% (IQR 8%-19.5%)](each P, <.05). Total cellularity (193,736/mL vs265,405/mL), leucocytes (46,378 vs 48,186), B (33 vs 76)and NK (215 vs 146) Ly events, % of B (0.1% vs 0.1%)and NK (0.2% vs 0.3%) Ly on leucocytes, % of T (96% vs93%), B (0.2% vs 1%), NK (1.4% vs 3.7%) Ly on total Lydid not differ significantly between cases and controls.BALF T Ly as events and as % of leukocytes indicate thesuspect of lung T-NHL, and the need of further deepenedFC analysis.

PROGNOSTIC VALUE OF FLOW CYTOMETRY EVAL-UATION OF BONE MARROW DYSPLASIA IN LOW-GRADE MYELODYSPLASTIC SYNDROMESTenore A.,1 Picone C.,1 Yokose N.,2 Matsuda A.,2Yamashita T.,2 Tamura H.,2 Tsukada J.,2 Dan K.M.,2Consensi E.,1 Vanelli L.,1 Ogata K.,2 and Della PortaM.G.11Dipartimento di Ematologia Oncologia, IRCCSPoliclinico San Matteo & Università di Pavia; 2Division ofHematology, Nippon Medical School, Tokyo, Japana.tenore@smatteo.pv.it

The current WHO classification of myelodysplastic syn-dromes (MDS) is based on morphological evaluation ofmarrow dysplasia. In clinical practice, the reproducibilityof the recognition of dysplasia is poor especially in caseswith low-grade disease that lack specific markers such asring sideroblasts and clonal cytogenetic abnormalities.We recently designed a flow cytometry score (FCM-score) applicable in many laboratories that may help toestablish the diagnosis of MDS (Haematologica.2012;97:1209-17). The FCM-score is based on four high-ly reproducible parameters analyzable from one cellaliquot, i.e. CD34+ myeloblasts (%), CD34+ B-cell pro-genitors (%), myeloblast CD45 expression, and channelnumber of side scatter where the maximum number ofgranulocytes occurs. In this multicentric study we aimed at evaluating the prog-nostic effect of FCM-score in a cohort of low-grade MDS.We studied 214 patients affected with refractory cytope-nia with unilineage dysplasia (n=58), refractory cytopeniawith multilineage dysplasia (n=133), sideroblastic anemia(n=18) and MDS with del5q (n=5). Patients were strati-fied according to International Prognostic Scoring System(IPSS). FCM-score value was 0 in 44 patients (21%), 1 in39 patients (18%), 2 in 69 patients (32%), 3 in 46 patients(21%) and 4 in 16 patients (7%). We confirmed a highinter-observer reproducibility of all cytometric parametersincluded in the score (CV ranging from 11 to 19%). AFCM-score ≥3 was significantly associated with the pres-ence of morphological multilineage dysplasia (p<.001),transfusion-dependency (P<.001) and unfavorable cyto-genetics (P=.007), leading to an higher IPSS risk

(P<.001). 5- year probability of survival was 74%, 65%and 17% in patients with FCM-score value <2, 2 and ≥3,respectively (P=.003), while 5-year probability ofleukemia-free survival was 89%, 78% and 47%, respec-tively (P=.004). In a multivariable analysis including age,sex and IPSS risk as covariates, FCM-score significantlyaffected the probability of overall and leukemia-free sur-vival (HR 1.42 and HR 1.56, respectively; P<.001). Thelikelihood ratios test (P<.001) showed that FCM-scoresignificantly improve the MDS prognostic stratificationprovided by IPSS. These results indicate thatimmunophenotyping based on four parameters with highinter-observer reproducibility may have important prog-nostic implications in low-grade MDS.

NEUTROPHILS ARE IMPAIRED IN MULTIPLE MYELO-MA BUT NOT IN MGUSTriolo A., Parrinello N. L., Rizzo G., Romano A., La Cava P.,Chiarenza A., Conticello C., Cavalli M., Palumbo G. A.,Di Raimondo F.Division of Hematology, Ospedale Ferrarotto, Catania, Italy cit.triolo@libero.it

Background: In Multiple Myeloma (MM) the immune func-tion is impaired but the role of microenvironment on thisdysfunction is unknown.Aims: Evaluation of immunosuppressive properties ofmature neutrophils in MM and MGUS.Methods: In 60 consecutive newly diagnosed MM, 70MGUS and 30 healthy subjects we evaluated in neu-trophils (N) from peripheral blood the phagocytic activityusing a commercially available kit (Phagotest R), and theexpression of molecule arginase-1 (Arg-1) and PROK-2.We tested the immunosuppressive properties with func-tional assay, based on in vitro co-culture of N isolatedfrom patients and T-lymphocytes from healthy subjects.Results: Despite no differences in the absolute number ofneutrophils between MM, MGUS and healthy donors, wefound a functional impairment in MM not evident inMGUS patients.The capability of phagocytosis of MM-N was significantlyreduced compared to healthy subjects (p<0.001) andMGUS (p<0.0001), and partially restored after inductionchemotherapy.MM-N exhibited an increased expression of ARG-1 com-pared to MGUS and healthy controls (25.5 vs 6.2 vs 1fold changes in gene expression, p=0.003), confirmed byfunctional assay of enzymatic activity of ARG-1, positive-ly correlated with advanced disease. Similarly, MM-Nexhibited an increased expression of PROK-2 comparedto MGUS and healthy controls (13.8 vs 1.5 vs 1 foldchanges in gene expression, p=0.001).After PHA-P stimulation, T-lymphocytes isolated fromhealthy donors missed the expression of activation mark-ers such CD71, CD69, CD25, CD3� in presence of MM-N for 72 hours, and in a less extensive way in presenceof MGUS-N.Summary / Conclusion: Compared to controls, neu-trophils obtained from MM patients and to a lesser extendfrom MGUS patients, have a reduced phagocytic activityand increased ability to suppress lymphocyte activation.These alterations may contribute to impairment ofimmune functions that characterizes MM patients.

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HUMAN AMNIOTIC EPITHELIAL CELLS EXPANDEDIN SERUM FREE MEDIUM MAINTAIN THEIRIMMUNOMODULATORY PROPERTIESAlviano F.,1 Ricci F.,2 Costa R.,1 Okere B.,3 Rossi M.,1Zannini C.,1 Simonazzi G.,4 Marchionni C.,1 Tazzari P.L.,2Bonsi L.,1 Pagliaro P.21Department of Experimental, Diagnostic and SpecialtyMedicine, University of Bologna, Italy; 2Immunohaematologyand Transfusion Medicine Service, S.Orsola-MalpighiHospital, University of Bologna, Italy; 3Department ofPaediatrics, University of Modena & Reggio Emilia, Italy;4Department of Medical and Surgery Sciences, University ofBologna, Italy francesco.alviano@unibo.it

Background: In the past years Amniotic Membrane (AM)has attracted great interest as available source of cellswith progenitor/stem cell potential and immunomodulato-ry properties. Clinical application of cellular therapiesrequires GMP-compliant culture condition: in this per-spective serum free culture systems are recommended.The aim of our study was to investigate if human AmnioticEpithelial Cells (hAECs) cultured in serum-free conditionpreserve their phenotypic and immunomodulatory fea-tures.Methods: hAECs were isolated from term placenta col-lected from caesarean sections and cultured in standardserum-rich medium (SRM) and serum-free optimizedmedium (SFM). Morphological observations and cell pro-liferation rate were assessed. We used flow cytometryNavios Beckman Coulter to evaluate immnophenotypefor stemness and epithelial markers expression. To studyin vitro immunomodulatory activity, hAEC were co-cul-tured on PHA-induced PBMCs in SRM or SFM. Theeffect of hAECs on PBMCs cell cycle and proliferationhas been analyzed through flow cytometer and BrdUincorporation. hAECs immune activity has been evaluat-ed also for tolerogenic molecule expression (i.e. HLA-G).Results: SFM sustained hAECs growth and stem cellpotential without any modification in morphology, cell pro-liferation rate and stemness. hAECs-mediated suppres-sion of activated PBMC proliferation was maintained inSFM: cell cycle analysis confirmed a marked reduction inpercentage of cells in S and G2/M phase as well as inSRM. Furthermore, BrdU incorporation rate of activatedPBMC decreased after co-culture with hAECs both withSFM and SRM. Finally, HLA-G expression increased sim-ilarly for hAECs cultured in SFM or SRM.Conclusions: AM holds great promise for the develop-ment of cell-based therapies in Regenerative andReparative Medicine. These data indicate that serum isnot essential for hAECs culture: serum-free culture con-ditions could simplify the transition from laboratory to clin-ical practice.

Immunology

HCV COINFECTION INTERFERENCE ON NK CELLPHENOTYPE AND FUNCTION IN VIROLOGICALLYHIV INFECTED SUPPRESSED PATIENTS TREATEDWITH MARAVIROCBozzano F1,2, Marras F3, Dentone C4, Parodi A2, DiBiagio A5, Cenderello G6, Orofino G7, Mantia E8, Guerra M9,Bartolacci V10, Fraccaro P11, Giacomini M11, Viscoli C12,Moretta L3, Filaci G1,2, Fenoglio D2, De Maria A2,5,13

1Dip. di Medicina Sperimentale-Università di Genova;2Centro di Eccellenza Ricerca Biomedica-Università diGenova;3 Istituto G. Gaslini, Genova; 4Osp. San Remo,Imperia; 5IRCCS AOU San Martino-IST Genova; 6Dip. diMalattie Infettive, Osp. Galliera, Genova; 7Dip. di MalattieInfettive, Osp. Amedeo di Savoia, Torino; 8Dip. di MalattieInfettive, Osp. SS. Antonio, Biagio, Cesare Arrigo,Alessandria; 9Dip. di Malattie Infettive, Osp. La Spezia;10Dip. di Malattie Infettive, Osp. Albenga; 11Dip. ofInformatica, Bioengineering, Robotica and SystemEngineering (DIBIS), Università di Genova; 12ClinicaMalattie Infettive, Az. Osp. Universitaria San Martino,Genova; 13Dip. di Scienze della Salute, Università diGenova, - Italyfederica.bozzano@unige.it

Background: Differences of activating NK cell receptorsat baseline are associated with the outcome ofpegIFNa+RBV treatment of chronic HCV infection (CHI).We here studied the relative contribution of HCV virus onNK cell phenotype and function in virologically HIV sup-pressed infected patients.Patients and Methods: NK cell phenotype and functionwere investigated by cytofluorimetry in 60 HIV infectedpatients. 36 HIV infected patients were treated withMaraviroc (MRV)-containing ART, while 24 patients wereon Maraviroc-free regimen (MRV-). 13/60 patients wereHCV coinfected. In HCV+MRV+ patients, the proportion ofCD56brightCD16+/- NK cell subset was increased com-pared to HCV+MRV- patients (*p<0,05), with comparableproportions of CD56dullCD16+ NK cells. CD56-CD16+ NKcells were lower in HCV+MRV- patients compared toHCV-MRV- (*p<0,05). NKp46 activating receptor expres-sion was reduced in HCV+MRV+ patients compared toHCV-MRV+ (*p<0,05). Expression of HLADR and CD69activation markers on NK cells were increased in HCV+coinfected patients compared to HCV-HIV+ patients(*p<0,05) and in MRV+HCV+ cohort of patients(*p<0,05). NK cell IFNgamma production was analysedby reverse ADCC against P815 cell line in presence ofmonoclonal antibodies anti-NKp46 and -NKp30.HCV+HIV+ coinfected patients showed reducedIFNgamma production compared to HCV-HIV+ infectedpatients. Conclusion: Differences in NK cell molecule expressionand function capability are present in HCV coinfectedpatients compared to HIV monoinfected patients and arenot treatment dependent.

EVALUATION OF THE DEGRANULATION PROCESSIN DIFFERENT NK CELL SUBSETS AFTER NKp46STIMULATIONBuccella F.,1 Del Zotto G.,1 Lucarini A.,1 CanonicoB.,1Luchetti F.,2 Arcangeletti M.,2 Papa S.,2 Zamai L.11Dipartimento di Scienze della Terra, della Vita edell’Ambiente, 2Dipartimento di Scienze Biomolecolari,Università degli Studi di Urbino “Carlo Bo”, Italy flavia.buccella@uniurb.it

Natural killer (NK) cells are large granular cytotoxic lym-

phocytes considered part of the innate immune system.They provide rapid responses to virally infected cells andtumor formation. Target cell recognition by NK cells is ahighly dynamic process controlled by the integration ofsignals from multiple receptors that promotes adhesion,cytokine secretion and/or granule polarization anddegranulation or inhibits NK cell functions. Among the dif-ferent NK activatory receptors, NKp46 (although its tumorligand is not unraveled) is considered one of the mostimportant ones in tumor recognition, but little is knownabout its trigger action in the different NK cell subsets.The aim of the present work was to define the synergy ofNKp46 stimulation with different co-activatory signals(2B4, DNAM-1, CD2) in the degranulation process of dif-ferent NK cell subsets. Experiments were performed atdifferent days after mononuclear cell isolation on restingor IL-2-cultured NK cells. In order to mimic the engage-ment of NK cell receptors with target cell ligands we usedbeads loaded with combinations of antibodies directagainst different NK receptors. To evaluate degranulatingNK cells, we have detected the surface expression ofCD107a (LAMP-1), a protein expressed on the inner sur-face of lytic granule membranes, with a specific mono-clonal antibody. NK cells were then stained with differentantibodies (anti-CD16, - CD56, -KIRs and -NKG2A) todistinguish CD56dim from CD56bright subsets or licensedfrom unlicensed NK cells. We observed that NKp46 pref-erentially sinergized with 2B4 stimulation on CD56dim

resting NK cell subset. As expected, the percentages ofdegranulating NK cells were significatively lower on unli-censed than on licensed ones in all stimulations. Thesedifferences tended be lost when NK cells were culturedfor 2-3 days with or without IL-2.

PERSISTENT INTRAHEPATIC Vγγ9Vδδ2 T-cells IMPAIR-MENT IN THE LIVER OF HCV-INFECTED PERSONS Cimini E.,1 Bordoni V.,1 Visco-Comandini U.,3 Montalbano M.,3Lionetti R.,3 Ricci F.,4 Bolognese A.,4 D’Offizi G.,3Capobianchi M.R.,2 Martini F.,1 Agrati C.1,2

1Cellular Immunology Laboratory, INMI-IRCCS “L.Spallanzani”; Rome; 2Virology Laboratory, INMI-IRCCS“L. Spallanzani”, Rome; 3Clinical Department, INMI-IRCCS “L. Spallanzani”, Rome; 4Surgery Department“Pietro Valdoni”, University “La Sapienza”, Rome.cimini.eleonora@libero.it

Hepatitis C virus (HCV) persistence in the host resultsfrom inefficiencies of both innate and adaptive immuneresponses to eradicate the infection. Among innateimmune cells, a functional impairment of peripheralVγ9Vδ2 T-cells was described during chronic HCV infec-tion; this functional defect can be partially restored byusing IFN-α, opening the possibility to boost antiviralinnate immune response. Unfortunately, no data areavailable on intrahepatic Vγ9Vδ2 T-cells that instead, mayrepresent the key actors in the anti-HCV response. Aim of our work was to analyze intrahepatic Vγ9Vδ2 T-cells in chronic HCV-infected patients (HCV) and, as acontrol, in healthy donors (HD) subjected to gut resectionsurgery. Phenotypic and functional features of intrahepat-ic and peripheral Vγ9Vδ2 T cells were analyzed in bothliver tissue and blood samples from 17 chronic HCVpatients and in 3 healthy control persons by flow cytome-try and ELISA assay.Irrespective of HCV infection, intrahepatic compartmentwas characterized by a lower frequency of Vγ9Vδ2 T cellsthan in the peripheral blood, showing an effec-tor/activated phenotype. In contrast, HCV infectioninduced an increase of PD-1 expression on both periph-

eral and intrahepatic Vγ9Vδ2 T-cells, probably due to per-sistent antigenic stimulation. Finally, liver residentVγ9Vδ2 T-cells showed a drastic reduction of their abilityto produce IFN-α both in HD than in HCV. Nevertheless,intrahepatic Vγ9Vδ2 T-cells from HD can be functionallyrestored by using IFN-α co-stimulation; in contrast a per-sistent Vγ9Vδ2 T-cell impairment was observed in theliver of HCV-infected persons. Interestingly, a studyaimed to evaluate the mechanisms of this process as wellas to find new pathways able to restore Vγ9Vδ2 T-cellsintrahepatic function may be mandatory.

CHARACTERIZATION OF IPEX AND ‘IPEX-like’SYNDROMES: FOXP3 AND RELATED PATHWAYCiullini Mannurita S.1, Vignoli M.1, Bacchetta R.2, Cecconi M.3,Tommasini A.4, Abinun M.5, Gennery A.5, Hambleton S.5,Slatter M.5, Holland S.M.6, Cant A. J.5, and Gambineri E.11Department of “NEUROFARBA”, section of Child’s Health,University of Florence and “Anna Meyer” Children’sHospital, Florence, Italy. 2San Raffaele Telethon Institute forGene Therapy (HSR-TIGET), Milan, Italy. 3GeneticLaboratories, Ospedale Galliera, Genoa, Italy. 4Departmentof Science of Reproduction and Development, University ofTrieste, Trieste, Italy. 5Department of Paediatric Immunology,Newcastle upon Tyne Hospitals, Newcastle upon Tyne, UK.6Laboratory of Clinical Infectious Diseases, National Instituteof Allergy and Infectious Diseases, National Institutes ofHealth, Bethesda.s.ciullini@meyer.it

IPEX (Immune dysregulation, polyendocrinopathy, endo-crinophathy, X-linked) is an autoimmune disorder charac-terized by severe early-onset enteropathy, endocrinopa-thy, dermatitis and autoimmune phenomena. It is causedby the alteration of the T regulatory cells function due tomutations in the FOXP3 gene. Not all patients with IPEXphenotype show FOXP3 gene mutations (IPEX-like syn-drome) and defects on CD25 and STAT5b have beenidentified in some of them.Overall, we collected 93 patients with suspect of IPEXsyndrome. Considering patients clinical features, Tregsand FOXP3 expression by flow cytometry, FOXP3, CD25and/or STAT5b genes were sequenced. FOXP3 mutations were identified in 16.1% of the casesand a variable FOXP3 expression by flow cytometry wasobserved. Low FOXP3 expression levels, compared withhealty subjects, where detected even in patients thatdon’t habour any alteration in the gene sequence. Absentexpression of CD25 was detected in two patients withCD25 mutations, confirming that protein expression ana-lysis can be useful to address the diagnosis in CD25 defi-ciency. One patient was characterized as a STAT5b defi-cency, moreover a STAT1 gain of function mutation cha-racterized by a prolonged phosphorylation of the proteinafter stimulation was recently identified in one patient. We confirm that flow cytometry is helpful in the characte-rization of patient with IPEX and IPEX-like syndromes. Inparticular we are developing the induced STAT phospho-rylation assay that will be a powerful tool both in the dif-ferential diagnosis with other primary immunodeficiencieswith immune dysregulation where STAT pathway defectsmight have a pathogenetic role and to explain the patho-genetic role of STAT genetic variation identified by mole-cular analysis.

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FOXP3+ AND CD14+ CELLS, TGF-BETA 1 LEVELS,BACTERIAL TRANSLOCATION INDEXES AND LIVERFIBROSIS SCORE: A CORRELATION STUDY INHIV+, HCV+, HIV+/HCV+ PATIENTS AND HEALTHYCONTROLSComolli G.,1,2 Sacchi P.,3 Corbella M.,3 Chiesa A.,2 Cima S.,3Calarota SA.,2 Baldanti F.,2 Marone P.,2 Novati S.,3Mulatto P.,3 Mariconti M.,3 Klersy C.,4 Baldi C.,5 Bruno R.,31Lab.Ric.Biotecnologie 2S.C. Microbiologia e Virologia 3Dip.Malattie Infettive 4Servizio Biometria e EpidemiologiaClinica. Fondazione IRCCS Policlinico San Matteo Pavia,Italy. 5Università degli Studi di Milano, Milano, Italyg.comolli@smatteo.pv.it

HIV infection seems to accelerate the progression of chro-nic C hepatitis. Regulatory T cell (Tregs) are CD4 + T cellsexpressing the forkhead-winged-helix transcription factor(Foxp3). Alterations in regulatory T cells (Treg) or in tran-sforming growth factor beta 1 (TGF-beta1) levels, as wellof bacterial traslocation markers might be involved in theaccelerated course of liver fibrosis seen in HIV ⁄HCV coin-fected individuals. A cross-sectional study was conductedon 80 subjects: HIV+ (n = 20), HCV+ (n = 20) andHIV/HCV-co-infected (n = 20) pts, and healthy controls (n= 20), older than 18 yrs. Flow cytometric % of Foxp3+ andCD14+ cells and TGF-beta 1 levels (by ELISA) were ana-lyzed in peripheral blood and correlated with liver fibrosis(measured either by biochemical score, FIB 4, or by elasto-metry). The correlation among: Foxp3+ and CD14+ cells,CD14 soluble levels, TGF-beta 1, IL17 and bacterial DNAproducts levels (expression of bacterial translocation) wasalso analyzed. The % of Foxp3+ cells was significant higherin HIV+ and HIV+/HIV+ pts than in both HCV+ pts and con-trol group (p< 0.0005, Kruskal-Wallis test). There was nostatistically significant difference for TGF-beta1 in the fourgroups. FIB 4 values inversely correlated with TGF-beta1(Rho correlation coefficient -0.38; p=0.0155), as well aswith liver stiffness values (Rho correlation coefficient -0.31;p=0.0498. CD14+ cells and CD14 serum levels were signi-ficantly different between HIV+ pts vs controls, HIV+/HCV+

pts vs controls, HCV+ pts vs HIV+/HCV+ pts (p< 0.0001,Kruskal-Wallis test). IL17 levels and bacterial DNA pro-ducts were significantly different between HCV+ pts vs theothers 3 groups. Foxp3+cells were higher in HIV+ pts, butthey did not influence liver fibrosis staging. TGF-b1 levelsinversely correlated with fibrosis suggesting a protectiveeffect. HIV+/HCV+ pts showed increased levels of bacterialDNA products, CD14 and IL17 expression as comparedwith the others groups. The correlation between the tran-slocation index and FIB4 suggest that fibrosis stage maybe related to an immunoactivation caused by bacterialtranslocation.

AIRE POLYMORPHISM, MELANOMA ANTIGEN-SPE-CIFIC T CELL IMMUNITY, AND SUSCEPTIBILITY TOMELANOMAConteduca G.,1 Fenoglio D.,1,2 Parodi A.,1 Battaglia F. ,1Kalli F.,1 Negrini S. ,1,2 Tardito S.,1 Ferrera F.,1 Salis A.,1Millo E.,1 Damonte G.,1 Indiveri F.,1,2 Ferrone S.,3Filaci G.1,2

1Excellence Centre for Biomedical Research, 2Departmentof Internal Medicine - University of Genoa, Genoa, Italy;3Department of Surgery, Massachusetts General Hospital,Harvard Medical School, Boston, MA, USAGiusy_Conteduca@libero.it

Background AIRE regulates T cell repertoire and is invol-ved in susceptibility to melanoma. In this study, we havetested the role mediated by melanoma antigen (MA)-

specific T cell immunity, taking advantage of the structu-ral and functional homology of human AIRE and MAGEwith their mouse counterparts. Methods AIRE and MAGEB2 expression was measuredby real time PCR in medullary thymic epithelial cells(mTECs) from two strains of C57BL/6 mice bearingselectively one of the two (T or C) allelic variants ofrs1800522 AIRE SNP. The apoptosis extent induced bymTECs in MAGEB2-specific T cells and the susceptibilityto in vivo melanoma B16F10 cell challenge were compa-red in the two mouse strains. Results The C allelic variant, protective in humansagainst melanoma, induced lower AIRE and MAGEB2expression in C57BL/6 mouse mTECs than the T allele.mTECs from the strain bearing the AIRE CC genotypeinduced lower extent of apoptosis in MAGEB2-specificsyngeneic T cells than mTECs from the strain bearing theTT genotype (P<0.05). Vaccination against MAGEB2induced higher frequency of MAGEB2-specific CTL andexerted higher protective effect against melanoma deve-lopment in mice bearing AIRE CC than in those bearingthe TT genotype (P<0.05).Conclusion These findings show that alternative allelicvariants of one AIRE SNP may differentially shape theMA-specific T cell repertoire potentially influencingsusceptibility to melanoma.

FLOW CYTOMETRIC EVALUATION OF HLA-GEXPRESSION IN CORD BLOODContini P., Ubezio G., Ghio M.Dept. of Internal Medicine - IRCCS Azienda OspedalieraUniversitaria San Martino-IST of Genoa, Italy.paola.contini@unige.it

Background: human umbilical cord blood (CB) is a usefulalternative to bone marrow (BM) and mobilized peripheralblood stem cells (PBSC) to rescue haematopoiesisfollowing myeloablative therapy. We hypothesize that apossible higher expression of HLA-G1 molecules mightplay a role in better host acceptance showed by cordblood transplantation. On this base, we analysed thecellular subsets expressing HLA-G, a well characterizedimmunoregulatory molecule able to inhibit innate andadoptive immunity. Materials and methods: to this aim, a flow cytometricevaluation (Beckman Coulter FC 500) of HLA-G1membrane expression was performed in 50 CB, 50peripheral blood (PB), 20 BM and 10 PBSC samples fromhealthy donors.Resukts: Total mean value of HLA-G1+ cells was 0.42%for CB compared to 0.04% for PB, 0.05% for PBSC and0.06% for BM samples, respectively. More in detail,CD34+ CD45+ HLA-G1+ cells in CB were 7.8% of totalcompared to 0.29% in PBSC and 0.05% in BM respec-tively. The HLA-G1+ stem cells were negative for themarkers of commitment. Finally, differing to what occursin the peripheral blood in which HLA-G1 is expressed byCD3+ cells, the further aliquots of HLA-G1+cellsdetectable in CB, PBSC and BM, were mainly CD19+.Conclusions: hematopoietic stem cells are endowed withpotent immunoregulatory activity. Even in partially HLA-mismatched setting and with a limited cellular dosage CBderived stem cells have been shown ability to engraft. Inour observations CB is characterized by a significanthigher percentage of HLA-G+ cells, compared to otherstem cells sources. HLA-G could be responsible of a“veto-activity” of CD34+ cells and the higher percentageof HLA-G+ cells in CB might explain a lower incidence ofHLA-mismatch related risk.

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INTERFERENCE OF HLA-DR STAINING ON CD124RECOGNITION IN THE IDENTIFICATION OFMYELOID-DERIVED SUPPRESSOR CELLS De Vita M.,1 Catzola V.,1 Battaglia A.,1 Buzzonetti A.,1Fossati M.,1 Zamai L.2 and Fattorossi A.11Lab. Immunol., Unità di Ginecol. Oncol., Univ. CattolicaS. Cuore, Roma; 2Dip. Scienze della Terra della Vita edell’Ambiente, Univ. Urbino ‘‘Carlo Bo’’, Italycytogyn@rm.unicatt.it

Myeloid-derived suppressor cells (MDSC) are immaturemyeloid cells that efficiently suppress T cell functions. Inthe setting of cancer immunology, MDSC are oftendefined by interleukin-4 receptor (CD124) expressionand low/absent HLA-DR expression by monocytes (MØ).During the immunomonitoring of clinical trials, weobserved large differences in the frequency of CD124+

MØ depending on different clones to the HLA-DRmolecule used. We show that different monoclonal anti-bodies (MoAb) that recognize different epitopes of theHLA-DR molecule (clones: L243, B8.12.2, G46-6, WR18,and 423L), variably interfere with the binding of anti-CD124 MoAb. The anti-HLA-DR clones G46-6 and WR18display higher interferences as compared with clonesL243 and B8.12.2, for the binding of anti-CD124 MoAb,and the anti-HLA-DR clone 423L does not interfere at allwith the binding. The anti-HLA-DR clone G46-6 inhibitedanti-CD124 MoAb binding irrespective of which MoAbwas added first. Inhibition was dose-dependent.Activating MØ in vitro with phorbol 12-myristate 13-acetate/ionomycin or phytohemagglutinin increasedCD124 and HLA-DR expression but did not prevent theinterference between the inhibitory clones of HLA-DRmolecule with the binding of anti-CD124 MoAb. Anti-CD124 MoAb binding to B cells, that express CD124 andHLA-DR, was inhibited by the various anti-HLA-DRMoAb, indicating that CD124 and HLA-DR molecules areclose each other, independent of the cell type. Theseobservations could have implications for both researchand clinical studies, in particular when dealing withimmunomonitoring of clinical trials.

INNATE IMMUNITY ACTIVATION IN VIROLOGICALLYSUPPRESSED HIV INFECTED MARAVIROC TREATEDPATIENTSDentone C.1,2, Parodi A.2, Bozzano F.2, Fraccaro P.4,Kalli F.2, Battaglia F.2, Marras F.2, Ferrea G.1, Viscoli C.5,Giacomini M.3, Filaci G.2,3, De Maria A.2,5, Di Biagio A.5,Fenoglio D.2,3 and MARHIV Study Group1Infectious Diseases Dept of Sanremo Hospital,2Excellence Centre for Biomedical Research (CEBR),3Dept of Internal Medicine, 4Dept of Communication,Computer and System sciences (DIBRIS), 5IRCSS,Infectious Diseases Dept, San Martino Hospital- Universityof Genoadaniela.fenoglio@unige.it

Background: The impact innate immunity status of differ-ent antiretroviral therapy (ART) protocols on innateimmunity needs to be elucidated. Methods: 68 virologically suppressed HIV patients inART ≥ 6 months were subdivided as taking (group G1) ornot (group G2) maraviroc (MVC). Circulating monocyteand natural killer (NK) cell cells were studied in correla-tion with clinical parameters. Results: Frequency of the inflammatory monocytes (iM)was lower in G1 than in G2 group. Decreased mean flu-orescence intensity (MFI) of CD38 and increased of PD-L1 were observed on these cells in the G1 group. The

HLA-DR+ NK cell frequency correlated positively withboth CD38 and HLA-DR MFI but negatively with PD-L1MFI on iM in the total patient population Several correla-tions between clinical and immunological parameterswere observed such as, in G1 group, a negative correla-tion between total cholesterol and the HLA-DR+NK cellpercentage and a positive correlation between minimumarterial pressure and HLA-DR MFI on iM. Conclusions: Frequency and activation of inflammatory sub-sets of innate immunity correlate with co-morbidity risk fac-tors in undetectable HIV-infected patients and are differentlycounteracted by cART protocols including or not MVC.

BEGEDINA®, CD26 AND GvHDDi Maria V., Tedone E., Dominietto A., Bacigalupo A.Centro Trapianto di Midollo Osseo, IRCCS San Martino-IST, Genova, Italy.valedima84@hotmail.it

Background. Allogeneic hematopoietic stem cell trans-plantation (HSCT) is the treatment of choice for themajority of hematological malignancies. Over the yearstransplant related mortality (TRM) was significantlyreduced due to better supportive care, improving HLA-typing and treatment of infections. However, mortality dueto steroids refractory acute graft versus host disease(aGvHD) remains high and unchanged. The aim of thisstudy is to evaluate the efficacy of BEGEDINA® in 12patients with steroids refractory aGvHD grade III-IV afterallogeneic HSCT in a pilot study (EUDRACT) and to testthe CD26+ expression on circulating CD3+ cells. Patients and methods.BEGEDINA® is a murine mono-clonal antibody against CD26 or Dipeptidyl peptidaseIV(DPPIV), an ubiquitous type II transmembrane glyco-protein expressed particularly on CD4+CD45RO+ mem-ory T cells, with binding activities.BEGEDINA® wasadministered at the dosage of 2 mg/day for 5 consecutivedays. Ten patients was grafted from unrelated donor, 2from HLA-ID sibling; the diagnosis was AL (7), MDS (2),NHL (2) and SAA (1). The expression of BEGEDINA®-CD26 on CD3+ cells was analyzed by flow cytometricevaluation (Beckman Coulter NAVIOS/FC500) on periph-eral blood samples, before and after each administrationof BEGEDINA®.Results.Ten patients (91%) showed a very goodresponse to the treatment with BEGEDINA®, 1PR and1NR. Despite the clinical results, the biological effect isnot so clear and the expression of BEGEDINA® CD26 onCD3+ cells was variable (25-90%) between patients andindependent from the clinical response.Conclusions.BEGEDINA® the murine monoclonal anti-body against CD26 in vivo is safety and efficacy for thetreatment of steroid refractory aGvHD. The expression ofCD26 on CD3+ cells is variable and is not related to theclinical response.

COMPARATIVE ANALYSIS OF CANCER VACCINESETTINGS FOR THE SELECTION OF AN EFFECTIVEPROTOCOL IN MICEKalli F.,1 Machiorlatti R.,2 Battaglia F.,1 Parodi A.,1Conteduca G.,1 Ferrera F.,1 Proietti M.,1 Tardito S.,1Sanguineti M.,3 Millo E.,1 Fenoglio D.,1,4 De Palma R.,5Inghirami G.,2 Filaci G.1,4

1Centre of Excellence, 4Dept of Internal Medicine-Univ ofGenoa; 2Dept of Pathology and Center for Exp Res andMedical Studies, Univ of Turin; 3IRCCS AziendaOspedaliera Universitaria San Martino IST, Genoa; 5Deptof Clin and Exp Medicine, Second Univ of Naples, Italyfrancesca.kalli@gmail.com

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Cancer vaccineshave not yet reached satisfactory clinicalresults, probably due to the difficulty to select an efficienttumor associated antigen and immunization protocol. Tothis issue, we performed a comparative analysis of differ-ent gp100 vaccination strategies to obtain protectionagainst experimental melanoma. C57BL/6J mice werechallenged subcutaneously with B16F10 melanoma cells,after vaccination with mouse or human gp10025-33 peptideplus CpG adjuvant; mouse or human gp100 gene; mouseor human gp10025-33 peptide-pulsed dendritic cells (DC).Alternatively, a neutralizing anti-IL10 monoclonal antibody(mAb) was subcutaneously administered at the site oftumor challenge to counteract regulatory cells. Finally,human gp10025-33 peptide-pulsed DC vaccination was per-formed associating with administration of the anti-IL10mAb. Vaccination with human gp10025-33 peptide-pulsedDC was the most effective immunization protocol, althoughnot achieving a full protection. Administration of the anti-IL10 mAb showed also a remarkable protective effect,replicated also in mice challenged with Anaplastic LargeCell Lymphoma. Combining gp10025-33 peptide-pulsed DCimmunization with IL10 counteraction, a 100% protectiveeffect was achieved. The analysis on tumor infiltrates Tcells showed increase of CD4+granzyme+ T cells anddecreased number of CD4+CD25+Foxp3+ Treg elementsfrom mice treated with either gp10025-33 peptide-pulsed DCvaccination or anti-IL10 mAb. These data suggest thatprocesses of intratumoral re-balance between effector andregulatory T cell subpopulations may play a critical protec-tive role in immunotherapy protocols.

ASSESSMENT OF DOPAMINERGIC RECEPTORS ONHUMAN CD4+ T CELL SUBSETS BY MEANS OF FIVE-COLOR FLOW CYTOMETRY ANALYSIS IN WHOLEBLOODKustrimovic N., Rasini E., Legnaro M., Dongmo B.,Marino F., Cosentino M. Center of Research in Medical Pharmacology, Universityof Insubria, Varese, Italynatasa.kustrimovic@uninsubria.it

Dopamine (DA) is a key neurotransmitters which acts onDA receptors (DR) classified, according to their pharma-cology and biochemistry, into D1-like (DRD1 and DRD5)and D2-like (DRD2, DRD3 and DRD4). Emerging evi-dence indicates that DA also exerts direct effects on theimmune system which are relevant in health and disease.Exploitation of the immune effects of DA requires howev-er better understanding of the pattern of expression of DRon immune cells.We developed a flow cytometric assay to examine the DRexpression on human CD4+ T lymphocyte subsets byuse of whole blood. Samples of venous blood wereobtained from healthy donors (F/M: 17/8; age[mean±SD]: 55.9±15.7 years). Immunophenotyping ofDR on CD4+ T cells was performed by a 5-color flowcytometric analysis in whole blood by use of a two-stepprotocol: during the first step, cells are stained for DR byan indirect labelling procedure (primary antibody (Ab) +secondary Ab, PE); during the second step, cells wereincubated with a cocktail of anti-human CD3, CD4,CD45RA and CCR7 ab conjugated with fluorochromes toidentify the subsets of interest. Expression of CD45RAand CCR7 allowed identifying the following subsets: Tcentral memory (Tcm, CD3+CD4+CD45RA-CCR7+), Tnaive (CD3+CD4+CD45RA+CCR7+), and T effectormemory (Tem, CD3+CD4+CD45RA-CCR7-).CD4+ T cells were (mean±SEM) 66.3±2.4% of total CD3+T cells. Among CD4+ T cells, Tcm were 28.7±1.9%, Tem

24.8±1.6% and T naive 43.4±2.7%. All five DR wereexpressed on CD4+ T cells. The D2-like DRD2, DRD3 andDRD4 were expressed by 3.1±0.5%, 6.6±1.1% and8.1±1.6%,, while the D1-like DRD1 and DRD5 wereexpressed by 11.7±1.4% and 13.2±1.2% of the cells(P<0.05 and P<0.0001 vs DRD2, DRD3 and DRD4). Thesame DR expression pattern was observed in CD4+ T cellsubsets, although statistical significance of the differencesbetween D2-like and D1-like DR was reached only in Tnaive cells (P<0.05 for DRD1 and P<0.0001 for DRD5).In conclusion, we developed a flow cytometric assay whichallows the straightforward analysis of DR on human CD4+T cell subsets. Knowledge of DR expression pattern onspecific subsets will provide a key contribution to planexperiments aimed at the understanding of the functionalrelevance of DR pathways in the regulation of the immuneresponse as well as to the interpretation of results. Thisstudy was supported by a grant from Fondazione CARIP-LO (Project 2011-0504: Dopaminergic modulation of CD4+T lymphocytes: relevance for neurodegeneration and neu-roprotection in Parkinson’s disease - The dopaminergicneuro-immune connection) to MC.

ANALYSIS OF CTLA-4 EXPRESSION ON PRIMARYMELANOMA CELL LINES AND THEIR REACTIVITYWITH IPILIMUMAB BY FLOW CYTOMETRYLaurent S.,1 Queirolo P.,2 Boero S.,3 Salvi S.,4 Boccardo S.,4Morabito A.,5 Fontana V.,6 Pietra G.,7 Carrega P.,8Ferrari N.,3 Mingari MC.,7 Poggi A.3 and Pistillo MP.51Department of Internal Medicine (DIMI), University ofGenoa, Italy - 2Department of Medical Oncology A, IRCCSAOU San Martino-IST, Genoa, Italy - 3Unit of MolecularOncology and Angiogenesis, IRCCS AOU San Martino-IST, Genoa, Italy - 4Department of Pathology, IRCCS AOUSan Martino-IST, Genoa, Italy - 5Unit of Tumor Geneticsand Epigenetics, IRCCS AOU San Martino-IST, Genoa,Italy - 6Unit of Epidemiology and Biostatistics, IRCCS AOUSan Martino-IST, Genoa, Italy - 7Department ofExperimental Medicine, University of Genoa, Genoa, Italy- 8G. Gaslini Institute, Genoa, Italylaurentstefania@libero.it

CTLA-4 (Cytotoxic T lymphocyte antigen-4) is a negativeregulator of T cell activation. The blocking of CTLA-4 withmonoclonal antibodies, such as Ipilimumab, is currentlyused in the treatment of metastatic melanoma to increaseantitumor immunity. Herein, we have analyzed CTLA-4expression and Ipilimumab reactivity on 17 melanomacell lines (14 primary and 3 long-term cell lines) by flowcytometry. Expression of surface CTLA-4 was analyzedby direct immunofluorescence using a PE-conjugatedgoat anti-CTLA-4 polyclonal antiserum (R&D Systems),whereas cytoplasmic CTLA-4 was analyzed by indirectimmunofluorescence using the unconjugated anti-CTLA-4 murine mAb (14D3 clone, from eBioscience). Next, wetested the reactivity of the therapeutic human mAbIpilimumab (Bristol-Myers-Squibb) both on the surfaceand in the cytoplasm of the melanoma cell lines followedby the addition of Alexafluor 647-conjugated goat anti-human IgG as secondary antibody (Molecular Probes,Inc.). Appropriate negative controls were included. Theflow cytometric analyses were performed with aFACSCalibur cytometer (Becton Dickinson) usingCellQuest software (Becton Dickinson). We found that allmelanoma cell lines, except FO-1, expressed variablelevels of surface and cytoplasmic CTLA-4 with both com-mercial and Ipilimumab antibodies. CTLA-4 expressionwas confirmed at trascriptional level by RT-PCR and byimmunohistochemistry on 33 melanoma tissues. We also

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observed that Ipilimumab can trigger innate immunity interms of antibody dependent cellular cytotoxicity (ADCC)or Tumor Necrosis Factor (TNF)-α release. Finally, axenograft murine model was set up to determine in vivothe effects of Ipilimumab and NK cells on melanoma. Ourstudies demonstrate that CTLA-4 is expressed not onlyon T cells but also on tumor cells and Ipilimumab triggerseffector lymphocytes to cytotoxicity and TNF-α release.These findings suggest that Ipilimumab can directly acti-vate the elimination of CTLA-4+ melanomas.

NUMERICAL ALTERATIONS OF DENDRITIC CELLSUBSETS IN THE PERIPHERAL CIRCULATION OFPATIENTS WITH PARTIAL 22q11.2 DELETIONSYNDROMELegitimo A.1, Terranova V.1, Orsini G1., Failli A.1,Caparello MC.1, Baroncelli GI.2, Consolini R.11Department of Clinical and Experimental Medicine,Laboratory of Immunology, University of Pisa, Pisa, Italy- 2Division of Pediatrics, Ospedale Santa Chiara – AOUP,Pisa, Italya.legitimo@ao-pisa.toscana.it

The immune deficiency of the 22q11.2 deletion syndrome(formerly called DiGeorge or velocardiofacial syndrome(DS)), is due to impaired development of the thymusgland; therefore, the majority of the studies have focusedon T cell numbers and function. Furthermore, a subgroupof patients with partial (p)DS appears to be at higher riskfor relevant infectious and autoimmune complications.The number and function of dendritic cells (DCs) in the22q11.2 DS has not been investigated, although thesecells represent the most important family of professionalantigen-presenting cells, able to efficiently prime naïve,memory and effector T cells and are responsible for acti-vating also natural killer T (NKT) cells.Therefore, we have enumerated circulating DCs in 13patients with partial 22q11.2 DS, directly from wholeblood using a four-colours flow cytometric method. DCsare also examined for the expression of CD85k antigenbecause it is down-regulated following DC activation andup-regulated in tolerogenic DCs, leading to the inductionof T reg cells.We report a significant decrease in the absolute numberof total DCs compared to age-matched healthy controls,which affects both myeloid and plasmacytoid DC (mDCand pDC) subsets.In particular, 9 out of 13 patients have a total DC numberunder -1SD of control mean. Interestingly, the patient withthe lowest mDC value suffered from metastatic thyroidcancer. The mean fluorescence intensity (MFI) of CD85kon mDCs is significantly higher in patients compared tocontrols, reaching in 7 patients values higher than +2SDof the mean of the controls.We suggest that the reduction of DCs could contribute tothe immunodeficiency of this syndrome; additional inves-tigations are necessary to dissect the heterogeneityobserved in this population in order to early detect thesubgroup of patients at higher risk of infections, tumoursand/or autoimmune disorders.

DISTINCTIVE FEATURES OF CLASSIC AND NON-CLASSIC (Th17 derived) HUMAN TH1 CELLSMaggi L., Santarlasci V., Capone M., Rossi M.C.,Querci V., Mazzoni A., Liotta F., Maggi E., Romagnani S.,Cosmi L., and Annunziato F.DENOTHE Center, University of Florence, Italylaura.maggi@unifi.it

T helper17 (Th17) lymphocytes represent a third arm of theCD4+ T-cell effector responses, in addition to Th1 and Th2cells. Th17 cells have been found to exhibit high plasticitybecause they rapidly shift into the Th1 phenotype in inflam-matory sites. In humans, Th1 cells derived from Th17 cellsexpress CD161, whereas classic Th1 cells do not; theseTh17-derived Th1 cells have been termed nonclassic Th1cells. In this study, we examined similarities and differencesbetween classic and nonclassic human Th1 cells by assess-ing a panel of T-cell clones, as well as CD161+ or CD161−CD4+ T cells derived ex-vivo from the circulation of healthysubjects or the synovial fluid of patients with juvenile idio-pathic arthritis. The results show that nonclassic Th1 cellscan be identified based on CD161 expression, as well as theconsistent expression of retinoic acid orphan receptor C, IL-17 receptor E, CCR6, and IL-4-induced gene 1, which are allvirtually absent in classic Th1 cells. The possibility to distin-guish these two-cell subsets by using such a panel of mark-ers may allow the opportunity to better establish the respec-tive pathogenic roles of classic and nonclassic (Th17derived) Th1 cells in different chronic inflammatory disor-ders.

THE IMMUNOSUPPRESSIVE NETWORK: CIRCULATINGMYELOID-DERIVED SUPPRESSOR AND T REGULATORYCELLS IN PATIENTS WITH NEOADJUVANT SHORT RTRECTAL CANCER PATIENTSNapolitano M.,1 Trotta A.,1 Cardone E.,2 D’Alterio C.,1Rega D.,2 Pace U.,2 Scala D.,2 Delrio P.,2 and Scala S.11Dipartimento di Immunologia oncologica; 2OncologiaChirurgica Colorettale, Istituto Nazionale Tumori,“Fondazione Giovanni Pascale”, Napoli, Italia;maria_napolitano@yahoo.it

Myeloid-derived suppressor cells (MDSCs) are a hetero-geneous family of myeloid cells that along T regulatorycells (Treg) play a major role in antitumor immuneresponse of cancer patients. In colorectal cancer patients,MDSCs have recently been described as correlating withstage, metastasis and chemotherapy respon se. A thera-peutic option for locally advanced rectal cancer (LARC) isneoadjuvant treatment based on short course radiothera-py based on 5 fractions of 5 Gy for 5 days (NRT). SinceMDSCs have been characterized as a population of cellsthat can regulate Treg cells, the overlapping target cellpopulation of Treg cells and MDSCs is indicative for theimportance and flexibility of immune suppression underpathological conditions. Aim of this work was to evaluatethe value of MDSCs and Treg cells in peripheral bloodLARC subjected to NRT and then surgery by MulticolorFlow Cytometric Analysis, to identify possible pattern ofimmune modulation. Thirteen patients were evaluated forboth circulating MDSCs and Treg cells s-I) at time 0, s-II)after two weeks from the beginning of RT), s-III) after 5weeks from the beginning of radiotherapy; s-IV) beforesurgery and s-V) 24 hours post surgery and s-VI) 6months after surgery s-VII) 12 months after surgery. Theanalysis of MDSC cells (identified as granulocytic LIN-

/HLA-DR-/CD33+/CD15+/CD11b+ and monocyticCD14+/HLA-DRlow-/CD11b+/CD33+) showed a reduction inall of 13 patients due to NRT, with a minimum level at pre-surgery sample (s-IV), more markedly in poor-responders6/13 (TRG III and IV) compared to responders patients7/13 (TRG I and II), this difference was statistically signif-icant (p = 0,037 repeated measure Anova Test); the num-ber of MDSC cells showed a recovery to baseline levels at12 months after surgery (s-VII) for all patients. The valueof Treg cells CD4+/CD25hi+/ FoxP3+ showed (although notsignificantly) a progressive strong increase in poor-

responder patients compared to the decrease in respon-ders patients, with the respective maximum and minimumpeaks to the pre-operative evaluation (s-V); after 12months after surgery (s-VII) these levels remain substan-tially different. Thus a clear immune response isdetectable in LARC according to NRT, it seems thatMDSC value could represent an early marker of responseto RT; moreover, an opposite trend between MDSC andTreg could be a useful indicator of poor prognosis.

FINGOLIMOD MODULATES PERIPHERAL EFFECTORAND REGULATORY T CELLS IN MS PATIENTSParodi A.,1 Serpero L.,1,2 Filaci G.,1,3 Battaglia F.,1

Kalli F.,1 Brogi D.,2 Mancardi GL.,1,2 Uccelli A.,1,2

Fenoglio D.1,3

1Excellence Center for Biomedical Research, Universityof Genova, Italy- 2Dep of Neuroscience, Rehabilitation,Ophthalmology, Genetics, Maternal and Child Health(DINOGMI) – University of Genoa, Italy - 3Dep of InternalMedicine, University of Genoa, Italyalessiaparodi@yahoo.it

Multiple sclerosis (MS) is a complex neurological diseasewhere, in genetically predisposed individuals, the unbal-anced interplay between pathogenic and regulatory Tcells will result in the progression of the autoimmuneassault to neural antigens. Fingolimod (FTY720), an oralsphingosine 1-phosphate modulator recently approvedfor the treatment of MS, inhibits the egress of T cells fromlymph nodes acting specifically on naïve and memory Tcells and sparing effector T cells. Here we characterizedIL-17 and IFN producing effector CD4 and CD8 positive Tcells as well as CD4 positive CD25highCD127low regulato-ry T cells in MS patients before and one month after treat-ment was started. We observed that did not significantlyaffect the percentage of CCR6 and CD161 positive Tcells in both CD4 and CD8 subsets, while it significantlyreduced the levels of both CD4+CCR6+CD161+ andCD8+CCR6+CD161+ producing IFN alone or in combi-nation with IL-17. The IL-17 secreting cell percentage inboth subsets was affected by the treatment to a lesserextent. Finally, we observed that CD4+CD25highCD127low

regulatory T cells were decreased in MS patients com-pared to healthy controls and significantly increased theirfrequencies. These findings demonstrate that FTY720functionally modulates the production relevant pro-inflam-matory cytokines in potentially pathogenic effector cellsand increases the number of circulating regulatory T cellspossibly contributing in restoring a balance betweenthese populations.

KIR2DS1-DEPENDENT ACQUISITION OF CCR7 ANDMIGRATORY PROPERTIES BY HUMAN NK CELLSINTERACTING WITH ALLOGENEIC HLA-C2+ DC OR TCELL BLASTSPesce S.1, Sivori S.1, Carlomagno S.1, Moretta L.2,Moretta A.1 and Marcenaro E.11Dipartimento di Medicina Sperimentale,Università degliStudi di Genova, Via L.B. Alberti 2, 16132 Italy; 2IstitutoGiannina Gaslini, Via G.Gaslini 5, 16147 Genova, Italysilvia.pesce@unige.it

Natural killer (NK) cells may capture the CCR7 chemoki-ne receptor from allogeneic CCR7+ cells by a mechanismtermed trogocytosis and acquire migrating properties inresponse to the CCL19/CCL21 lymph node chemokines.This event is negatively regulated by inhibitory KIRs andNKG2A. In this study, we analyzed the role of activatingKIRs in the process of CCR7 uptake in NK cells interact-

ing with different types of allogeneic CCR7+ cells.Analysis of freshly isolated peripheral blood NK cells andNK cell clones revealed that in KIR2DS1+ alloreactive NKcells, co-cultured with HLA-C2+ CCR7+ lymphoblastoidcell lines, the interaction of KIR2DS1 with HLA-C2 (its lig-and) resulted in increased CCR7 uptake. We show thatKIR2DS1 represents a remarkable advantage for theacquisition of CCR7 also from HLA-C2+ allogeneic DCand T cell blasts because of its ability to override inhibi-tion by NKG2A. These findings have important implica-tions in haploidentical haematopoietic stem cell trans-plantation in which donor-derived (alloreactive)KIR2DS1+ NK cells upon acquisition of CCR7 becomeable to migrate towards lymph nodes, where they may killpatient DCs and T cells, preventing graft-versus-host andhost-versus-graft reactions.

NEW IMMUNOASSAY TO MEASURE INTESTINALAUTO-ANTIBODIESQuaglia S.1, De Leo2 L., Granzotto M.3, Petix V.2, Di LeoMG.2, Martelossi S.2, Ventura A.2, Not T.2,

1

1University of Trieste, Trieste, Italy; 2Department ofReproductive, Developmental and Public HealthSciences, Institute of Child Health, I.R.C.C.S. “BurloGarofolo”, Trieste, Italy; 3Laboratory Department A.O.U.Ospedali Riuniti, Trieste, Italy

In coeliac disease (CD) anti-tissue transglutaminaseautoantibodies (anti-tTG) were synthesized in the intesti-nal mucosa, even when absent in serum, and can beused in the diagnosis of CD especially for those serone-gative subjects with high risk for CD.The aim of our study was to develop a new operator-inde-pendent assay to measure intestinal anti-tTG in compari-son with the double coloured immunofluorescence stai-ning in detecting the IgA anti-tTG deposits on the intesti-nal biopsies.We enrolled 153 consecutive patients who underwentupper gastrointestinal endoscopy at Children’s Hospital.Two biopsies were obtained: one for double colouredimmunofluorescence staining and one for acid antibodyextraction. The eluted samples were buffered and quan-tified for total protein concentration. 50µg of proteins fromeach sample were incubated with magnetic beads coatedwith human tissue transglutaminase to capture anti-tTGantibodies. Beads were incubated with anti-human IgAFITC and analyzed by flow cytometry. The flow cytometrycut off value was calculated as the media ± one standarddeviation of the percentage of fluorescent beads obtainedfrom CD negative intestinal samples.We calculated the cut off value of 15%. We found 84 posi-tive samples (mean 77±22 s.d.) and 69 negative results(mean 2,3 ±3,4 s.d.). In double coloured immunofluore-scent assay we had 85 positive results and 68 negativeones. In comparison with the latter assay the beadsassay had a sensitivity of 95% and a specificity of 95%.Among the study population there were 5 asymptomaticsubjects with no serological markers, without any intesti-nal damage who were both positive for IgA anti-tTG inte-stinal deposits in double coloration fluorescence assayand beads assay. These patients were put to gluten freediet.Our preliminary results suggest that beads assay is analternative operator-independent assay to measure anti-tTG deposited in the intestinal mucosa also in the veryearly stage of CD. This assay can also be adapted todetect other autoantibodies in any tissue samples.

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EVALUATION OF LYMPHOCYTE ACTIVATION MARK-ERS, POST-THYMIC NAIVE AND MEMORY T CELLSSUBSETS AND HLA LOCI B,Cw FOR THE MONITOR-ING OF HIV INFECTION IN THE ACUTE PHASERicci M.1, DiGiuli C.2, Adami C.1, Palmieri G.1,Scaccetti A.1 e Materazzi M.1

1Immunoematology and Blood Trasfusion Unit, FlowCytometry Laboratory S.Maria Terni Hospital; 2Dept. ofMedicine, Infectious Diseases Ward S.Maria Terni Hospitaland University of Perugia School of Medicine, Italymonia_ricci@virgilio.it

Many authors agree on the need, to identify additionalcytofluorimetrics markers beyond CD4+ and CD8+ sub-sets of T cells, for assessing lymphocyte activation andevaluate post-thymic naïve and memory cell subsetsexpression, in naïve HIV patients undergoing currenttreatments. It has been previously shown the ability ofalleles B57-58, B27 and Bw4 to mediate a CD8+ cytotox-ic control mechanism in HIV-positive patients, usingCD38, HLA-DR and CD45RO. In this study, we evaluat-ed activated CD4+ and CD8+/HLA-DR+/CD38+ cells,post-thymic naïve CD4+ and CD8+/CD45RA+/CD62L+cells and memory CD4+ and CD8+/CD45RA+/CD62L-cells on naïve HIV-positive patients both before and afterstarting HAART therapy.12 HIV+ blood samples fromInfectious Diseases ward and 12 blood samples fromdonors at Terni Blood Bank were studied usingFacsCANTOII BD at T0 and T1, T2 and T3 post HAARTtreatment. They were incubated with TBNK (BD),CD8FITC, CD38PE, CD3PerCP, HLA-DR APC as activa-tion markers, with CD45RA-FITC, CD62L-PE,CD3PerCP, CD8APC for naïve and memory cells, andanalysed using the Canto and DIVA software programs.For serological typing of HLA, we used Biotest plates andDynal Magnetic Beads. 5 HIV naïve patients showed highlevels of CD8/CD38/HLA-DR and of naïve and memorycells. In those patients a decrease in HIV-RNA wasachieved wich did not correlate with a decreaseCD8/CD38/HLA-DR and naïve and memory cells levels.This was probably due, as stated in the literature, to HBV,HCV and TBC coinfection, confirming persistent T cellsactivation. The remaining 7 HIV+ patients, showed areduction in CD38/CD8 levels of expression and CD4 andCD8 memory cells, as expected after HAART terapy, withsimilar trends at 4-8-12 months post treatment (stastical-ly significant p=0,025 of Student Tests). Patient 5 withhaplotype B35 and CW4 showed a depletion of CD4 andCD8, of naive post-tymyc and memory T cells subsetsand activation markers. Patients with alleles B57 andBw4 showed a response to therapy with lowering ofviremia and a persistent cytotoxic activation mechanism.

COMBINATION THERAPY WITH IPILIMUMAB ANDELECTROCHEMOTHERAPY (ECT): PRELIMINARYEFFICACY RESULTS AND CORRELATION WITH TREGULATORY CELLS (Treg)Simeone E.1, Benedetto L.2, Gentilcore G.1, Capone M.1,Madonna G.1, Caracò C.2, Di Monta G.2, Marone U.2, DiCecilia M.2, Grimaldi A.M.1, Mori S.2, Mozzillo N.3 andAscierto P.A1. 1INT Fondazione “G. Pascale”- Unit of Melanoma,Cancer Immunotherapy and Innovative Therapy, Naples,Italy; 2INT Fondazione “G. Pascale”- Division ofMelanoma, Sarcoma and Skin Cancer, Naples, Italy; 3INTFondazione “G. Pascale” - Department of Melanoma,Sarcoma and Skin Cancer, Naples, 80131, Italymarilenacapone@gmail.com

Background: Ipilimumab is the first agent approved forthe treatment of advanced melanoma that showed a sur-vival benefit in randomized phase 3 trials. It is associatedwith a slow onset and low rate of responses and, in manycases, responses occur after other therapies, like target-ed therapy and radiotherapy. ECT has been shown to beeffective for treatment of cutaneous and subcutaneousmelanoma metastases. The current challenge is toimprove Ipilimumab efficacy in combination/sequencewith ECT to verify the possible increase of response rate.Furthermore, we evaluated the T-Reg variations inperipheral blood mononuclear cells (PBMC) of treatedpatients.Methods: We analyzed data from 10 patients (pts) withadvanced melanoma (6 stage IIIc and 4 stage IV M1c).They were treated with ipilimumab at 3 mg/kg every 3weeks for 4 cycles (day 1) and ECT with bleomycine at15 mg/m2 (day 2) on superficial lesions. PBMCs werecollected on day 0, 1 and 2, then on day 15 and 30 fromECT, at each cycle of ipilimumab and at every tumor eval-uation (every 12 weeks). T-Reg were analyzed by FACSanalisys labeling PBMCs with anti-CD4, CD25 and anti-FoxP3.Results: 10/10 (100%) pts showed local objectiveresponses (4 CR and 6 PR). 7/10 (70%) pts showed localresponse (6 PR and 1 CR) after the second ipilimumabdose and 3/10 showed response (3 CR) at week 12. Twopts with PR (28%) out of the group of 7 showed responseon distant lesions at week 24 (abscopal effect). To date allpts are still alive with a median follow up of 11 months(range 6-18). In all pts a decrease of T-Reg of 0.10%(range 0.50-2.6%) per cycle and no variation of CD4+and CD25+ lymphocytes has been observed.Conclusion: The preliminary results show that the combi-nation approach with Ipilimumab and ECT could improvethe responses compared to the Ipilimumab treatmentalone. The decrease of circulating T-Reg cells could beassociated with better response to treatment. Furtherstudies about this combination are warranted.

CORRELATION BETWEEN FLC AND CYTOMETRICEVALUATION OF PLASMACELLS IN MONOCLONALGAMMAPATHY AND IN PATIENTS WITH MULTIPLEMYELOMA: A CLINICAL CASE EXEMPLARY Tommasino C., Fossati G., Bloise A., Carbone C.,Russo A., Insidioso M. U.O.C. Patologia Clinica - P.O. S.Gennaro - ASL Na 1Centro Italyc.tommasino@libero.it

BACKGROUND. Flow cytometry and immunologicalmethods have allowed the characterization of a series ofsurface antigenic molecules expressed on either MM ornormal cells. With this techniques several moleculesdifferentially expressed on normal and MM cells andcorrelated with the prognosis of MM patients have beenidentified. Normally, the light chains are produced inexcess and therefore a fraction is present in the serum infree form (FLCs). In monoclonal gammapathy, theproduction of FLC type k o λ exceeds the normalconcentrations also altering the ratio κ / λ (rFLC).MATERIALS AND METHODS.We have studied a series

of 60 patients with monoclonal gammopathy(40 MGUSand 20 MM)and followed for about 6 months with periodicevaluations of ESP, IFE, FLC assays and bone marrowaspirate. Among our data there is also a difference(dFLC) between free light chains involved in thepathology (iFLC) and uninvolved free light chains (uFLC),which is important in monitoring patients in therapy.The

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BIOCOMPATIBILITY ANALYSIS OF NOVEL (butylene1,4-cyclohexanedicarboxylate) BASED ELECTRO-SPUN COPOLYESTERS CONTAINING PEG-likeSEQUENCES FOR TISSUE ENGINEERING APPLICA-TIONSBloise N.,1 Amoroso FC.,1 Monaco A.,1 Gigli M.2,Gualandi C.,3Focarete M.F.,3 Mazzini G.,4 Lotti N.,2 Visai L.,1,5

1Dept. of Molecular Medicine, UdR INSTM, and Ctr forTissue Engineering (CIT), Univ. Pavia; 2Dept. of Civil,Chemical, Environmental and Materials Eng., 3Dept ofChemistry ‘‘G. Ciamician’’, UdR INSTM, Univ. Bologna,;4IGM-CNR and Dep. of Biology & Biotehnologies, Univ.Pavia,; 5Dept. of Occupational Medicine, Ergonomy andDisability, S. Maugeri Foundation, IRCCS, Pavia, Italynora.bloise@unipv.it

Tissue engineering aims to regenerate damaged tissues,instead of replacing them, by developing biological substi-tutes that restore, maintain or improve tissue function. Itrelies extensively on the use of porous 3D-scaffolds toprovide the appropriate environment for the regenerationof tissues and organs. The scaffolds, which essentially actas a template for tissue formation, were produced from avariety of biomaterials and manufactured using a plethoraof fabrication techniques. Among these last, electrospin-ning is the only method capable of producing continuouspolymer nanofibres. Electrospinning is a unique technolo-gy that can produce non-woven fibrous articles with fiberdiameters ranging from tens of nanometers to microns.We propose a new class of aliphatic polyesters based onpoly(butylene 1,4-cyclohexandicarboxylate) (PBCE). Inparticular, random copolymers containing PEG-glycolsubunits have been prepared, fully characterized and sub-jected to electrospinning in order obtain fibrous scaffoldsto be used in tissue engineering applications. In this study,we aimed at investigating skeletal myogenesis on thesenovel electrospun scaffolds. The potential effects of alltested copolymers were evaluated by investigating C2C12proliferation (via CCK8 assay) and differentiation (viaMHC immunostaining). We demonstrated that they enableskeletal myogenesis in vitro by aiding in (a) myoblastadhesion and proliferation, and (b) myotube alignment, byproviding the necessary directional cues along with archi-tectural and mechanical support. Further studies will beconduct in order to demonstrate their application in the tis-sue engineering field.

IN VITRO COMPARATIVE STUDY OF THE EFFECTSOF ZINC-CONTAINING BIOACTIVE GLASS-COATEDTITANIUM ON PROLIFERATION AND OSTEOGENICDIFFERENTIATION OF HUMAN MESENCHYMALSTEM CELLS Bloise N.,1,4 Risi G.,2 Vercellino M.,1,4 Quartarone E.,2,4

Mustarelli P.,2,4 Mazzini G.,3 Ingo D.,6 Avanzini M.A.,6Imbriani M.,5,7 Visai L.,1,4.1Dept. of Molecular Medicine, 2Dept. of Chemistry, 3IGM-CNR and Dept. of Biology & Bioteh UniPV, 4Ctr for TissueEngineering (C.I.T.), 5Dept.of Public Health, ExperimentalMedicine and Forensic, UniPV Pavia,; 6PediatricOncohematology Lab., Policlinico San Matteo Foundation,IRCCS, Pavia,; 7Dept. of Occupational Medicine, Ergonomyand Disability, S. Maugeri Foundation, IRCCS, Pavia, Italynora.bloise@unipv.it

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immunophenotype of bone marrow plasmacellsdemonstrated that 10/40 MGUS were CD56 positivewhile 20/20 MM were CD56 positive. We haveextrapolated the case specimen of a seventy patientwhose MM was diagnosed in November 2011.RESULTS AND CONCLUSIONS. In the patientconsidered we have observed an excellent therapeuticresponse through the densitometric evaluation of the CMin association with the assay of serum FLC, whichcorrelates directly with the number of bone marrowplasma cells. In correspondence of the increases in dFLCbone marrow plasmacells increase in number andincrease the positivity of CD56 on their surface.As expected, the plasmacell movement is directly relatedto the increase in dFLC. This assay allows however tomonitor in a reliable, sensitive and certainly less invasiveway than bone marrow aspirate the trend of the disease.This assay allows to reduce the number of IFE and bonemarrow aspirates reserving these investigations to theoccasions in which the dFLC increases.

LONG-TERM WHOLE BLOOD DONATION CANAFFECT THE BALANCE BETWEEN NAIVE ANDMEMORY T LYMPHOCYTE POPULATIONS IN HUMANPERIPHERAL BLOODUrbani S., Amadei B., Portararo G., Tanzi F.,Formentini A., Fagnoni F.F.UO di Immunoematologia e Medicina Trasfusionale,Dipartimento di Patologia e Medicina di Laboratorio,Azienda Ospedaliero-Universitaria di Parma, Italyffagnoni@ao.pr.it

To provide protection against both unknown new patho-gens and recurrence of previous infections, the immunesystem maintains adequate populations of naive andmemory T cells, respectively. However, thymic output ofnaïve T cells is drastically reduced in human adults. Wehypothesized that long-term frequent donation of wholeblood could constitute a model of chronic T cell depletion.Thereby, we asked whether such cell loss may have animpact on the naive T cell reservoir and peripheral T cellsubsets homeostasis. To evaluate the composition of cir-culating T lymphocyte in relationship to high numbers ofdonations, 55 donors with a median age of 58 who haddonated >85 times were enrolled in this study along with55 age-matched donors who had donated <15 times. Thestudy has been performed by flow cytometry on samplesof red-cell lysed whole blood labeled by staining withCD3, CD8, CD28, and CD95 antibodies. Stained sam-ples have been acquired through a FACSCalibur flowcytometer and analyzed by CellQuest Software.Percentages of naïve T cells in peripheral blood of long-term donors were significantly lower than in donors whodonated few times. In particular, in long-term donors,naive (CD95-CD28+) T cells represented 22% (± 7.4 SD)of gated CD3+ T cells, whereas in donors who donatedfew times they represented 36% (± 11.2 SD) (p<0.001,Mann-Whitney test). Conversely, effector memory(CD95+CD28-) T cells were significantly more abundantin long-term donors (27% ± 12.5) as compared to occa-sional donors (15% ± 9.7) (p<0.001). By contrast, centralmemory T cells (CD28+CD95+) did not differ between thetwo groups. Differences between the two donors’ groupswere more pronounced within the CD8+ than the CD4+ Tcell subset. Results from this preliminary study suggestthat an high number of donations of whole blood canreduce the proportion of the naïve T cell reservoir in peri-pheral blood while homeostasis of T cells is maintainedthrough expansion of effector memory T cells.

Methodology and Technology

Bioactive glasses coating of titanium (Ti) scaffolds arevery attractive materials for bone regeneration due totheir versatile properties. An important feature of bioglassit that, upon implantation, its surface undergoes complexkinetic modifications leading to the formation of an amor-phous calcium phosphate layer. Bioactive glasses can beimproved with trace quantities of elements such as Cu,Zn and Sr, which are known to be beneficial for healthybone growth. The aim of this study was to in vitro evalu-ate the biological reactivity of bioglass coating titaniumcontaining 0.4% wt ZnO in terms of hMSC proliferationand differentiation. The mesenchymal stem cells isolatedfrom adult human bone marrow (hBMSC) or from humanadipose tissue (hASC) was seeded on scaffolds. MTTassay was performed to assess the biocompatibility ofthe scaffolds while the distribution of the cells inside theconstructs was analyzed by Scanning ElectronMicroscopy (SEM). The osteo-inductive potential of thesescaffolds was investigate by the immunolocalization ofosteogenic markers (i.e. Alkaline phosphatase, ALP)using Confocal Laser Scanning microscopy (CLSM). Incomparison with uncoating titanium and bioglass coated3D Ti 58S, the 58S-Zn0.4 bioglass-coated 3D Ti scaffoldshowed a significant increase in cellular proliferation.SEM analysis showed that cells more homogenouslycovered the surface of 58S-Zn0.4 disk than to 58S anduncoating titanium disks. As shown by CLSM, ALP pro-duction was higher with respect to titanium and 58S. Weconclude that the treatment of titanium surface with bio-glass and 0.4%wt zinc oxide, improves its ability toenhances mesenchymal stem cells adhesion, prolifera-tion and their osteogenic differentiation.

COMPARISON BETWEEN WESTERN BLOT ANDFLOW CYTOMETRY IN THE DETECTION OF PTENAND mTOR IN PEDIATRIC T-CELL ACUTELYMPHOBLASTIC LEUKEMIABonaccorso P.1,2, Bugarin C.1, Fazio G.1, LoNigro L.2,Cazzaniga G.1, Biondi A.1 and Gaipa G.1.1Pediatric Clinic, University of Milan Bicocca,M.Tettamanti Research Center, Monza, Italy2Center of Pediatric Hematology Oncology, AziendaPoliclinico - OVE, Catania, Italypaolabonaccorso@libero.it

Signalling networks such as the PI3K-AKT-mTOR path-way play a role in the modulation of the aggressivenessof T-Cell Acute Lymphoblastic Leukemia (T-ALL), but theprognostic significance of an aberrant activation of thispathway is poorly investigated. Western Blot (WB) repre-sents the method of references to study proteomic profilein hematological malignancies, but Flow Cytometry (FC)can also be applicable at single-cell level in the same set-ting (Irish JM et al, Nat Rev Cancer 2006), however thechoice of the best method can be still an open question.We analyzed the PTEN and mTOR basal expression inbone marrow (BM) samples from 9 children with T-ALLand 9 normal subjects1}. Western blot analysis was per-formed using standard RIPA buffer for protein extraction.A StripAblot Stripping Buffer was used to recover mem-branes. Ratios between target protein over beta-actinhave been calculated by densitometry analyses, usingAlliance instrument and Uviband software. Sampleswere examined also by FC as previously described(Gaipa G et al, Leukemia 2008), and protein levels weremeasured as % of positive cells compared to isotype con-trol. Positivity or negativity by WB was established bypresence or absence of protein band, while for FC thresh-old for positivity was set at ≥ 1% of positive cells.

Qualitative concordance (positive Vs negative) was100% using the two methods, being 3/ 9 and 2/9 T-ALLsamples PTEN and mTOR negative, respectively. Meanvalues of PTEN/mTOR ratio in T-ALLs was highly concor-dant when measured by FC or WB: 5.40 and 5.57respectively, and this result was highly reproducible whenindividual paired samples (n=4) were considered. Ninenormal BM samples were also analyzed by FC: 7/9 and9/9 were PTEN and mTOR-positive, respectyively.Interestingly PTEN was significantly higher in T-ALLs ascompared to normal BM: 47.00%±26.81% Vs4.30%±2.89% (p=0.001), by contrast mTOR was higherin normal BM as compared to T-ALLs: 8.70%±3.54% Vs29.70%±30.22% (p=0.06), consequently PTEN/mTORratio resulted reversed in the two subgroups: 5.40 in T-ALLs and 0.14 in normal BM samples. Moreover, whenonly CD7+ T lymphocytes (instead of total nucleatedcells) were analyzed in normal BMs 9/9 were PTEN neg-ative by FC (<1,0%), while in T-ALL CD7+/CD45 lowblasts resulted either PTEN negative (3/9) or overexpressed (6/9, mean PTEN positive cells: 47.00%,range 18.4%-90.5%). Our results showed that WB andFC techniques provide full concordant qualitative (posi-tive Vs negative) read outs in the measurement of bothPTEN and mTOR in T-ALLs, and highly concordantPTEN/mTOR ratio measurements. Of note, PTEN/mTORratio in normal BM and in T-ALLs are inversely correlatedand this observation was not previously reported, sug-gesting further investigations.

EVALUATION OF DIELECTROPHORESIS FORISOLATION OF PANCREATIC ISLETS FROMEXOCRINE TISSUEBurgarella S.,1 Merlo S.,2 Figliuzzi M.,3 Remuzzi A.3,4

1STMicroelectronics, Advanced System Technology,Agrate Brianza, Milan, Italy. 2Dipartimento di IngegneriaIndustriale e dell’Informazione, University of Pavia,Pavia, Italy. 3Mario Negri Institute, c/o Parco ScientificoTecnologico Kilometro Rosso, Bergamo, Italy.4Department of Engineering, University of Bergamo,Dalmine, Bergamo, Italysarah.burgarella@st.com

Dielectrophoresis (DEP) has been reported as a promis-ing method for cell manipulation without physical contactin miniaturized lab-on-chip devices, since it exploits thedielectric properties of cells suspended in a fluid and sub-jected to high-gradient electric fields. Objective of thiswork is to demonstrate the applicability of DEP for thepurification of Langerhans islets from fragments of pan-creatic exocrine tissue, a critical stage for the furthertransplantation of insulin secreting islets in patientsaffected by type I diabetes. A critical aspect of the appli-cation of this physical phenomenon to the manipulation ofpancreatic islets is related to their size. DEP is usuallyapplied for the manipulation of single cells, with dimen-sions of the order of microns, while Langerhans islets areaggregates of cells and have a minimum diameter of 100µm. Dielectric models of single pancreatic beta cells andLangerhans islets as cell aggregates were developed.Numerical simulations were performed to optimize thegeometric design of the microelectrodes configuration aswell as to determine the applied electric field and theforces subsequently acting on pancreatic islets andexocrine tissue fragments. A custom electronic setup wasdeveloped for the generation of sinusoidal signals withproper voltage and frequency. Experiments on developedelectrodes configurations were performed with singlepancreatic beta cells and with samples of Langerhans

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islets and pancreatic exocrine tissue fragments. Dielectricmodels of cells and islets were verified and negative DEP,showing repulsion from the electrodes, was observed forpancreatic islets. The results of this work demonstrate thatLangerhans islet cells can be manipulated without physicalcontact by dielectrophoresis, a technique which can beapplied on single cells as well as on cell aggregates inminiaturized lab-on-a-chip devices.

MULTIPARAMETRIC FLOW CYTOMETRY OF BRON-CHOALVEOLAR LAVAGE FLUID CELL ANALYSIS BYSINGLE TUBE TEN COLOR Ciriello M.M.1, Bensi T.1, Calcagno L.1, Coppo M.1,Vasta M.1, Polla B.2, Arfini C.1

S.S. Antonio E Biagio E C. Arrigo Hospital, 1Laboratory ofClinical Pathology, Departement of Services, 2RespiratoryDisease Unit, Departement of Internal Medicine.Alessandria, Italymciriello@ospedale.al.it

Bronchoalveolar lavage (BAL) analysis in flow cytometry(FCM) is an important diagnostic tool in various diffuselung diseases. Respect conventional immunochemistry,FCM immunophenotyping of lymphocytes in BAL sam-ples provides results with excellent sensitivity and speci-ficity. Using FCM BAL analysis CD4/CD8 ratio determina-tion is reliable, precise and fast. Inflammatory cell differ-ential counts in BAL can also be easily performed by mul-tiparametric FCM, rather than with cytology on cytospinpreparations, that is more time consuming and greathervariability affected. The clinical utility of bronchoalveolarlavage fluid (BAL) cell analysis for the diagnosis andmanagement of patients with interstitial lung disease(ILD) has been recently discussed by a committee ofinternational experts of American Thoracic Society, butthere are not FCM guidelines about antibody panels andgate strategies. Between 2006 and 2012 we have performed more than200 BAL fluid analysis by FCM and, from the last year, weperform them with a ten color multiparametric staining ina single tube, using Navios Cytometer (Beckman Coulter)and Kaluza software for analysis. To determine cell differ-ential count we performe BAL cytospin by opticalmicroscopy and BAL FCM labelling to detect cell differen-tiation and lymphocyte subsets. Usually we use a panelwith monoclonal fluorochrome conjugated antibodiesdirected against CD3, CD4, CD8, CD9, CD14, CD15,CD19, CD45, CD(16+56) and HLA-DR. There is a goodcorrelation between FCM and optical microscopy for dif-ferential cell count, although when it is performed by cyto-centrifuge technique underestimates lymphocytes, partic-ularly if their numbers are high. The technology develop-ment we allowed to improve the FCM method for BAL cellanalysis. Our results show that FCM is feasible and reli-able method to count BAL cell fraction based on cell anti-gen expression.

ANALYSIS OF CIRCULATING ENDOTHELIAL CELLS(CECs) AND ENDOTHELIAL PROGENITORS (EPCs)BY FLOW CYTOMETRY FOR CLINICAL PURPOSES:URGENT NEED OF A METHODOLOGICALSTANDARDIZATIONDanova M.1, Manzoni M.2, Comolli G.3, Torchio M.1,Gristina V.1, Mazzini G.41Medicina Interna e Oncologia, Ospedale di VIGEVANO, A.O.di Pavia, 2Oncologia Medica, Ospedale di CREMA, 3Laboratoridi Ricerca Biotecnologie, Fondazione IRCCS S. Matteo, PAVIA,4Istituto Genetica Molecolare C.N.R., PAVIA Italymarco_danova@ospedali.pavia.it

Given the importance of vasculature in the biology oftumors (as well as in several diseases), also as apotential target for treatment, there is an increasing needfor biomarkers of angiogenesis. Flow cytometric (FCM)detection of CECs and EPCs in whole PB is a fast assayto enumerate and characterize these cells in clinicalstudies, but remains a technical challenge. Their numberis low and they cannot be characterized by a singlemarker but often discriminated using a combination ofantigen with low, dull or a continuum of cell surfaceexpression. We have evaluated 20 clinical studies published from2007 to 2013, based on the utilization of FCM. Eachpaper was analyzed for: FCM protocol, panels ofendothelial markers, type of disease, number of pts andstudy design, overall clinical value ascribed to CECs andEPCs. Herein we summarized the pertinent findingswhich likely contributed to the differences observedacross studies. The multitude of detection FCM methodsapplied (not always sufficiently detailed), togheter withthe heterogeneity of the pt series, greatly limitscomparability. A lack of a inter-laboratory standardizedFCM assay and of a consensus on CEC and EPCphenotype has resulted in an enormous heterogeneity inthe reported number of CECs and EPCs. Hence, evenwhen looking at similar clinical conditions, the overallclinical value of the studies is partially limited.In conclusion, although significant changes in the numberof CECs and EPCs in different diseases have beenreported and several steps forward the definition of thepotential for clinical purposes have been achieved, theirreliable identification and quantification still remainsdifficult. For clinical purposes, the interpretation of resultsshould be made cautiously; the specificity of the antigeniccombination for CEC and EPC definition and the inter-laboratories standardization of enumeration proceduresare highly warranted to use these cells as clinicalbiomarkers.

A NOVEL APPROACH TO DETECTING RNA EXPRES-SION IN LIVING BREAST CANCER CELLS Weldon D., Su K., Ko A., Johnston G., Hsu M., Armstrong L.and Guarino G.*Merck Millipore Corporation – Temecula – USgoffredo.guarino@merckgroup.com

When studying the complexities of cancer biomarkers,the ability to monitor gene expression within live cells thathave become cancerous provides better insight thanexamining fixed or lysed tissues. However, monitoringRNA within intact cells can prove to be challenging withcurrent techniques, which involve complex RNA isolationprocedures that compromise cell viability. Further, ampli-fication methods can create false positives or erroneous-ly inflate differences. Here we describe a probe-baseddetection method that allows for the detection of ErbB-2and EGFR mRNA levels across live intact SK-BR-3breast cancer cells without the need for transfectionreagents. The detection methodology is based upon anoligonucleotide gold nanoparticle conjugate capable ofdetecting intracellular levels of mRNA and miRNA in liv-ing cells. The probes enter the cell by means of the cell’snative endocytosis machinery. Upon binding to the com-plementary target RNA sequence, a fluorescent signal(flare) is released, which allows the cells to be detectedon any fluorescence analysis platform. In contrast to tra-ditional RNA detection methods, since our techniqueallows for detection within live cells without altering theirgene expression, the same cells that were profiled for

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EGFR and ErbB-2 expression could be sorted based ontheir specific mRNA content and subsequently furtherstudied. Stimulation or treatments could then be per-formed on the previously profiled cells and assessedusing probes for additional RNA markers or more tradi-tional techniques such as antibody staining, qRT-PCR, orarray profiling. For this purpose, we combined immunos-taining for cell surface EGFR with fluorogenic nanoparti-cle-based detection of EGFR mRNA in four breast cancercell lines known to range in EGFR expression levels, andwe observed significant correlation between mRNA andprotein signals using two-color flow cytometry. Thus,detecting RNA expression levels in live cancer cells withthe ability to utilize the same cells in downstream testinggives researchers the ability to perform experimentswhich were previously thought to be impossible.

GROUP I METABOTROPIC GLUTAMATE AUTORE-CEPTORS INDUCE ABNORMAL GLUTAMATE EXO-CYTOSIS IN A MOUSE MODEL OF AMYOTROPHICLATERAL SCLEROSISGiribaldi F.1, Milanese M.1, Bonifacino T.1, Rossi P.2,3,Di Prisco S.1, Pittaluga A.1,4, Tacchetti C.1, Puliti A.2,3,4,Usai C.5, Bonanno G.1,4.1Dept. Exp. Medicine, Univ. of Genoa, Italy; 2Mol.Genetics and Cytogenetics Unit, Gaslini Inst., Genoa,Italy; 3Dept. Pediatric Sci., Univ. of Genoa, Italy; 4Centerof Excellence for Biomedical Research, Univ. of Genoa,Italy; 5Inst. of Biophysics, CNR, Genoa, Italyusai@ge.ibf.cnr.it

Glutamate-mediated excitotoxicity plays a major role inALS and reduced astrocytic glutamate transport was sug-gested as a cause. Based on previous work we have pro-posed that abnormal release may represent another sour-ce of excessive glutamate. In this line, here we studied themodulation of glutamate release in ALS by Group I meta-botropic glutamate (mGlu) receptors, that comprise mGlu1and mGlu5 members. Synaptosomes from the lumbar spi-nal cord of SOD1/G93A mice, a widely used murine modelfor human ALS, and controls were used in release, confo-cal or electron microscopy and Western blot experiments.Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG>0.3 µM stimulated the release of [3H]d- aspartate, used tolabel the releasing pools of glutamate, both in control andSOD1/G93A mice. At variance, ≤0.3 µM 3,5-DHPG increa-sed [3H]d-aspartate release in SOD1/G93A mice only.Experiments with selective antagonists indicated the invol-vement of both mGlu1 and mGlu5 receptors, mGlu5 beingpreferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 for-mation in both mouse strains, whereas low 3,5-DHPG didit in SOD1/G93A mice only. Release experiments confir-med that 3,5-DHPG elicited [3H]d-aspartate exocytosisinvolving intra-terminal Ca2+ release through IP3-sensitivechannels. Confocal microscopy indicated the co-existenceof both receptors presynaptically in the same glutamater-gic nerve terminal in SOD1/G93A mice. To conclude, acti-vation of mGlu1/5 receptors produced abnormal glutamaterelease in SOD1/G93A mice, suggesting that these recep-tors are implicated in ALS and that selective antagonistsmay be predicted for new therapeutic approaches.

PERFORMANCE EVALUATION OF A SIMPLE FLOWCYTOMETER IN HEMATOPOIETIC STEM CELLCOUNT: A COMPARISON WITH A GOLD STANDARDINSTRUMENTMariani M., Colombo F., Cattaneo A., Assennato S.M.,Frugoni C., Rebulla P., Porretti L. Centro Interdipartimentale di Citometria, FondazioneIRCCS “Ca’ Granda” Ospedale Maggiore Policlinico,Milano, Italylabcitofl@policlinico.mi.it

Accurate estimation of cell counts by flow cytometry maybe difficult in laboratories where sophisticated equipmentand staff with specific flow cytometry expertise are notavailable. Relatively inexpensive Flow Cytometers (FC)able to perform basic functions may help to overcomethese difficulties.In this study we compared Hematopoietic Stem Cell (HSC)and leukocyte counts determined with the small and rela-tively inexpensive BD Accuri® C6 FC equipped with CFlowPlus® v. 1.0 software, with those obtained with the eight-color BD FACSCantoII® equipped with FACSDiva® v. 4.0.2software, our gold standard instrument.We tested in parallel 85 consecutive samples from cordblood, peripheral blood from onco-hematology patients,fresh and thawed HSC collected by apheresis and bonemarrow products. All samples were analysed in duplicate.Results were compared with the one way ANOVA test,linear regression and Bland-Altman analysis. Resultreproducibility was expressed as coefficient of variation(CV) of replicates. According to our procedures, CVexceeding 20% require test repetition.Time for instrument setup, calibration and analysis wasslightly longer with Accuri C6® (40 min) than withFACSCantoII® (30 min). Calibration of Accuri® C6 failed inabout 20% of the experiments; a second calibration requiredfurther 15 minutes. Both HSC and leukocyte counts werehighly correlated (r2>0.99; P<0.0001) and did not differ sig-nificantly (p>0.05); the Bland–Altman analysis showed goodcount concordance with a moderate trend to larger deviationat low HSC counts (0-10/µl). Mean CVs of HSC and leuko-cyte count replicates were 7.1% and 3.3% for the Accuri® C6and 4.9% and 3.1% for the FACSCantoII®, respectively, withno cases exceeding 20%.In conclusion, Accuri® C6 is a reliable instrument for HSCand leukocyte enumeration. Its use is very simple anddoes not require deep flow cytometry knowledge. Its per-formance may be improved by a more efficient calibrationand shorter analysis time.

3D CELL MICROINCUBATOR WITH INTRINSIC OPTI-CAL TRANSDUCTION CAPABILITY FOR ADVANCEDCELL ANALYSESMerlo S.,1 Carpignano F.,1 Silva G.,1 Surdo S.,2 Barillaro G.,2Aredia F.,3 Scovassi A.I.,3 and Mazzini G.3,4

1Dipartimento di Ingegneria Industriale e dell’Informazione,Università di Pavia, Pavia, Italy. 2Dipartimento di Ingegneriadell’Informazione, Università di Pisa, Italy. 3Istituto diGenetica Molecolare, CNR, Pavia, Italy. 4Dipartimento diBiologia e Biotecnologie “L. Spallanzani”, Università diPavia, Pavia, Italysabina.merlo@unipv.it

We have demonstrated that vertical high aspect-ratiophotonic crystals can play the role of microincubators forcell culture in a three-dimensional (3D) environment andsimultaneously the role of label-free optical detectors.The investigated microstructures, fabricated by electro-chemical micromachining of standard silicon wafers, con-

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sist in periodic arrays of 3 µm thick silicon walls separat-ed by 5 µm wide, deeply etched air-gaps and feature thetypical spectral properties of photonic crystals in the nearinfrared wavelength range 1.0 µm – 1.7 µm. Their spec-tral reflectivity, detected by means of a light beam travel-ing back and forth through silicon walls and gaps, is char-acterized by wavelength regions where propagation isforbidden and reflectivity is high (photonic bandgaps),separated by narrow regions where reflectivity is verylow. As the presence of cells grown inside the gapsstrongly affects light propagation across the photoniccrystal and its spectral reflectivity, we have exploited thisfeature to directly probe the cell distribution with a label-free optical detection method. In particular, after demon-strating that human fibrosarcoma HT1080 cells are ableto actively populate the deep, narrow gaps, growingadherent to the vertical silicon walls, we have used afiberoptic setup for spatially mapping the variations of theline-shape of the reflected power spectrum due to the cellpresence. Finally, we have shown that the intensity ratioat two specific wavelengths is well correlated to the cellspreading onto the silicon wall. These results representthe starting point for future in-situ label-free analyses ofcellular activities (e.g., proliferation, differentiation andapoptosis) involving changes in cell morphology and/oradhesion, directly performed on a microstructure suitablefor 3D cell culture.Work partially funded by FAMT, Pavia, Italy, andFondazione CARIPLO.

MESENCHYMAL STEM CELLS: BIOPHYSICAL CHAR-ACTERIZATION AND OSTEOGENIC DIFFEREN -TIATION Petecchia L.,1 Vercellino M.,2 Visai L.,2 Usai C.,1

Sbrana F.,1 Vassalli M.,1 Gavazzo P.1

1Institute of Biophysics, National Research Council,Genoa, 2Department of Molecular Medicine and Center forTissue Engineering (C.I.T.), University of Pavia, Pavia, Italygavazzo@ge.ibf.cnr.it

Human mesenchymal stem cells (hMSC) are able to selfrenew or to be committed towards cells of various lineag-es, such as osteoblasts. For this reason they haveacquired a promising role in the field of regenerative med-icine, even if major limitations to their clinical applicationto repair bone defects exist due to the poor availability ofcells and the time required to differentiate to a stage suit-able for implantation. Physical stimuli, such as pulsedelectromagnetic fields (PEMFs), have been widely usedin orthopedics and, at the moment, PEMF therapy isapproved for bone disorders. The mechanism throughwhich PEMF promotes the formation of bone remainshowever elusive. Our activity focused on the in vitro char-acterization of the molecular mediators of the effects ofPEMF treatment on osteogenic differentiation of hMSC.In particular, through a combined biochemical and bio-physical approach, we concentrated on the role of calci-um, highlighting a potential role of intracellular calciumstores in the PEMF-induced differentiation pathway.

DRUG DELIVERY SYSTEMS: MULTI-WALLEDCARBON NANOTUBES AS CARRIERS FORMITOXANTRONERisi G.,1 Bloise N.,2 Merli D.,1 Profumo A.,1 Fagnoni M.,1Mustarelli P.,1 Imbriani M.,3,5 Mazzini G.,4 Visai L.2,5.1Dep. of Chemistry, 2Dep. of Molecular Medicine andCenter for Tissue Engineering (C.I.T.), 3Dep.of PublicHealth, Exp. Medicine and Forensic, 4IGM-CNR and Dep.of Biol. & Biotech. Univ. Pavia; 5Dep. of OccupationalMedicine, Ergonomy and Disability, S. MaugeriFoundation, IRCCS, Pavia, Italygiulia.risi02@ateneopv.it

Carbon nanotubes (CNTs) have been introduced recent-ly as a novel carrier system for both small and large ther-apeutic molecules. Most of the research on CNTs hasfocused on their potential for delivery of anticanceragents. In our work will discuss the efficacy ofMitoxantrone (antineoplastic drug, MTX) absorbed onmultiwalled-CNT (MWCNTs) on human breast cancer cellline, MDA. MTX was adsorbed on oxidized MWCNTs byinteractions with CNT carboxylic group and drug aminegroups. The adduct has been characterized throughRaman Spectroscopy, Thermo Gravimetric Analysis(TGA), and TEM. Trypan blue dye exclusion assay wasused to investigate the cytotoxic effect of MTX adsorbedon CNTs. The results showed that Mitoxantrone loadingon carbon nanotubes produced a reduction in MDA cellviability slightly higher if compared to MTX in solution.Cell viability in the presence of MTX loaded carbon nan-otubes was dose- and time-dependent. These prelimi-nary results showed that MWCNTs-MTX are as mucheffective as the free drug in killing tumor cell line; itsphysico-chemical and pharmacokinetics properties maysupport its use as an in-situ neo-adjuvant and/or adjuvantcytotoxic device. Actually, we are studying the combina-tory effect of MWCNTs-MTX and some antineoplastic gal-lium salts, with the purpose of obtain a reduction of ther-apeutic MTX dose, maintaining drug’s efficacy.

NEW SOURCE OF MULTIPOTENT MESENCHYMALCELLS FOR TISSUE ENGINEERING STUDIES:WASTE HUMAN OVARIAN FOLLICULAR FLUIDRiva F.1, Omes C.2, Fassina L.3, Vaghi P.4, Reguzzoni M.5,Mazzini G.6, Casasco A.11Dip. Sanità Pubblica, Medicina Sperimentale eForense, Unità di Istologia ed Embriologia generale,Università di Pavia; 2IRCCS Fondazione Policlinico S.Matteo di Pavia, Centro di Procreazione MedicalmenteAssistita, Ostetricia e Ginecologia; 3Dip. di IngegneriaIndustriale e dell’Informazione, Università di Pavia,Centro Ingegneria Tissutale; 4Centro GrandiStrumenti, Università di Pavia; 5Dip. ScienzeChirurgiche e Morfologiche, Università dell’Insubria,6IGM-CNR e Dipartimento di Biologia e Biotecnologie,Università di Pavia, Italyfederica.riva01@unipv.it

3D biomaterials have been widely used in reconstructivebone surgery as cell carriers providing mechanical sup-port and as promoters of cell attachment and prolifera-tion. In particular, gelatine cryogel scaffolds are promisingnew biomaterials owing to their biocompatibility and tosubstain the differentiation of mesenchymal stromal stemcells (MSCs). MSCs are multipotent stem cells that prolif-erate onto the surfaces with fibroblastic morphology andcan differentiate into osteoblasts, chondrocytes andadipocytes. These cells can be isolated from severalsources, including bone marrow and adipose tissue. Our

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previously studies showed the possibility to obtain MSCsalso from the human ovarian follicular liquid (FL) that isusually wasted during in vitro fertilization. In this study,our aim was to show the possibility to use FL cells com-bined to a promising biomaterials for tissue engineeringin regenerative medicine. We tested the ability of theseFL cells to grow and differentiate in minimal culture con-ditions on gelatine cryogel, in comparison with MSCsderived from human bone marrow. Samples and controlswere analyzed with confocal and scanning electronmicroscopes. Results showed that mesenchymal FL cellson gelatine cryogel scaffold were able to adapt to surfacestructure of biomaterial scaffold, growing not only on thetop surface but also in the layers below till 60 µm of deep-ness. Positive immunostaining for vimentin and CD44 (atypical MSC marker) of these cells on cryogel confirmedtheir mesenchymal stemness and demonstrated anexcellent compatibility with the biomaterial surface. Bonemarrow MSCs, used as control, showed a similar behav-ior. Preliminary results showed also the capability of FLcells on scaffold to be induced to osteogenic differentia-tion, producing bone extracellular matrix and expressingsome specific proteins (i.e.osteopontin). In conclusion,MSCs derived from waste human ovarian follicular liquidshowed promising affinity with 3D gelatine cryogel, open-ing new potential developments in biotech and medicalapplications.

MULTICOLOR IMMUNOFLUORESCENCE STAININGALLOW DETAILED MORPHOLOGICAL STUDIES OFA549 LUNG ADENOCARCINOMA CELLS AFTERTGF-ββ1 TREATMENTSessa G., Camerlingo R., Irollo E., Miceli R. and Pirozzi G.Cancer Stem Cell Unit – Research Department I.N.T.-IRCCS “G.Pascale” of Naples, Italyg.sessa@istitutotumori.na.it

Objectives: Here we investigate Cytoketatin (CK) andVimentin (Vm) markers distribution in A549 lung adeno-carcinoma cell line before and after TGF-β1 treatmentand we propose our immunofluorecent staining protocol.Methods: To investigate CK and Vm expressions, cellswere plated in a 24well plate (P24w) in which roundslides to allow the cells to grow above were placed. At70% of confluence, cells were treated with 2ng/ml ofTGF-β1 for 48 hours. Then, untreated and treated cellswere fixed in 4% paraformaldehyde for 20 min at +4°Cand permeabilized with 0.5% Triton for 5 min. A blockingstep was performed with 5% BSA. The primary antibod-ies dilution was 1:150 for rabbit anti-Human Cytokeratin(Abcam) and 1:100 for mouse anti-Human Vimentin(Abcam), overnight at +4°C. The secondary antibodiesdiluition was 1:400 for goat anti-rabbit IgG DyLight594(Abcam) and 1:500 for goat anti-mouse IgG FITC(ImmunoReagents, Inc.), for 90 min. at +4°C. The nucleiwere counterstained with DAPI 300nM (LONZA).Multicolor staining were performed as following: DAPI-CK, DAPI-Vm, DAPI-CK-Vm. Isotype controls were usedfor each sample.The samples were observed with Axioplan2 equippedwith a HBO100 mercury vapor lamp and an AxioCamMRc5 camera, and analyzed using AxioVision Rel.4.8.2software, equipped with the module MultidimensionalAcquisition, all provided by Zeiss.Results: The untreated and treated A549 cells expressedboth CK and Vm markers. In untreated cells, IF analysesshowed a homogeneous distribution of CK whereas Vmwas localized in perinuclear vesicles in cytoplasm. Aftertreatment, IF assay showed down-regulation of CK and

Vm resulted to be distributed homogeneously to formintermediate filaments of cytoskeleton. Discussion: Despite the problems in the multicolor stain-ing protocol as the possible crossreactivity between theantibodies used, this staining allows you to make anaccurate analysis of the distribution of makers analyzedon the same cell.The simultaneous staining with the two markers in asso-ciation with DAPI counterstaining, allows to appreciatethe different localization of the markers around the nucle-us of the cells in order to better understand the effect ofTGF-β1, the main inducer of Epithelial-MesenchymalTransition process, on remodeling of cytoskeleton.

A COMPARISON BETWEEN OPERATOR ANDAUTOMATED SOFTWARE ANALYSIS OF FLOWCYTOMETRY DATA Sizzano F.1, Valle A.2, Di Terlizzi S.1, Palini A.3, Monti P.2,Villa C.11Flow cytometry Resource Advanced CytometryTechnical Applications Laboratory (FRACTAL), SanRaffaele Scientific Institute, Milan, Italy; 2DiabetesResearch Institute, San Raffaele Scientific Institute,Milan, Italy; 3Flow Cytometry Unit, Nestlè Institute ofHealth Sciences, Lausanne Switzerlandsizzano.federico@hsr.it

In the recent years, several software packages for theautomated analysis of flow cytometry data have becomeavailable. Here, we aimed to compare the traditionaloperator-dependent analysis of T-cell subpopulations onmultiple samples with the analysis performed by theSpanning-tree Progression Analysis of Density-normal-ized Events software (SPADE, Qiu et al., Nat. Biotech2011, Bendall et al. Science 2011). Whole blood collect-ed from 3 healthy donors was stained with fluorophore-conjugated monoclonal antibodies against CD3, CD4,CD8, CCR7 and CD45RA. After red blood cell lysis, sam-ples were acquired and T-cell subpopulations were inde-pendently determined by an operator (using computer-assisted tools) or by means of SPADE. The data,expressed as the percentage of cells in the parent gate,were analyzed using statistics for method comparison.When all measurements were pooled, results showed agood agreement between the two methods: (bias: 0.8; r:0,97; linear regression: y=0,6229+0,9949x). When thesingle T-cell subpopulations were analyzed, biasobtained was: total CD4: 0.4; CD4 naïve (n): 1.9; CD4central memory (cm): 0.5; CD4 effector memory (em): -0.43. For CD8 T-cells bias was: total CD8: 0.4; CD8n: 0.2;CD8cm: 5.2; CD8em: 2.7; CD8emRA: -3.4. Again, theseresults showed a good agreement between the two meth-ods for all T-cell subpopulations, except for CD8cm andCD8 emRA. This discrepancy could be due to the lownumber of measurements as well as the continuous andnon distinct pattern of the two subpopulations. Our pre-liminary results suggest that the automated analysis isconsistent with the operator-based approach. The imple-mentation of the automated method could support routinecytometry data analysis, thus providing a robust and sig-nificantly more rapid means of extrapolating multi-dimen-sional data in multi-sample (longitudinal) studies.

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HUMAN PLACENTA-DERIVED NEUROSPHERES ARESUSCEPTIBLE TO TRANSFORMATION AFTEREXTENSIVE IN VITRO EXPANSIONAmendola D.1, Nardella M.2, Guglielmi L.2, D’Agnano I.2,Stigliano A.3, Novelli G.1, Bucci B.11Research Center San Pietro Hospital Fatebenefratelli,Rome, Italy; 2CNR-Institute of Cell Biology andNeurobiology, Rome, Italy; 3Endocrinology Department ofClinical and Molecular Medicine, Sant’Andrea Hospital,Faculty of Medicine and Psychology, Rome, Italydonatellaamendola@yahoo.it

Introduction: The cancer stem cell model links neoplasticcells with normal stem cell biology, but little is known onhow normal stem cells are transformed into cancer stemcells. Methods: To investigate the processes underlying thetransformation of normal stem cells we developed in vitroa cancer stem cell model from human amniotic and cho-rionic placenta membranes. In this model we studied theexpression of specific stem cell molecules by flow cyto-metry, and genes by real time RT-PCR. Microscopyimmunfluorescence was employed to investigate the pro-liferative and differentiation patterns. Paired t-test analy-sis was employed for the statistical significance.Results: Normal human stem cells from amniotic andchorionic placenta membranes differentiated into neuralcell lineages, under specific conditions, to form seconda-ry neurospheres with a capacity for self-renewal. Afterextensive in vitro culture, these cells underwent sponta-neous transformations and acquired a neuroblastoma(NB)-like phenotype with an elevated proliferative poten-tial that is comparable to established neuroblastoma celllines. The ability of these cells to transform their phenoty-pe was evidenced by an increased clonogenic ability invitro; by augmented expression level of certain prolifera-tion- and transformation-related genes (e.g., CCNA2,MYCN, ENPP2, GRIA3, and KIT); by the presence ofmultinucleated and hyperdiploid cells. We further demon-strate that the transformed phenotype is an NB by mea-suring the expression of an NB-specific cell-surface mar-ker, disialoganglioside GD2. Conclusions: We have developed a cancer stem cellmodel starting from normal human stem cells derivedfrom amniotic and chorionic placenta membranes. Thesecells are able to differentiate into neural cell lineages andto undergo spontaneous transformations and acquire anNB-like phenotype.

EVIDENCES OF IMMUNOFLUORESCENCE ASSAYSUSING FLUORESCENT SILICA NANOPARTICLES(NPs) BIOCONJUGATEDCalabrese V.,1 Rossi E.,2 Facchinetti A.,2 Menna M.,1Zamarchi R.,3 Juris R.,11AcZon Srl, Via Lavino 265-D, 40050 Monte San Pietro,Bologna, Italy-www.aczonpharma.com - 2Department ofSurgery, Oncology and Gastroenterology, OncologySection, University of Padova, Via Gattamelata 64,35128 Padova, Italy - 3IOV-IRCCS, Via Gattamelata 64,35128 Padova, Italy valentinacalabrese@aczonpharma.com

Fluorescent antibodies are widely employed in many

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A SIMPLE METHOD FOR THE TITRATION OF THEISOTYPE-MATCHED ANTIBODY COULD BE USEFULFOR A BETTER CONTROL OF THE BACKGROUNDFLUORESCENCESizzano F.,1 Di Terlizzi S.,1 Canonico E.,1 Muradore I.,1Palini A.,2 Villa C.11Flow cytometry Resource Advanced CytometryTechnical Applications Laboratory (FRACTAL), SanRaffaele Scientific Institute, Milan, Italy. 2Flow CytometryUnit, Nestlè Institute of Health Sciences, Lausanne,Switzerlandsizzano.federico@hsr.it

The precise definition of the boundary between the posi-tive (+ve) and the negative (-ve) cell population for thoseantigens displaying a continuos pattern of expression,remains one of the most discussed issues in flow cytom-etry. Several methods are used to minimize and controlthe background fluorescence to better define the +vepopulation, including antibody (Ab) titration,Fluorescence Minus-One controls (FMO) and Isotype-matched Controls (IC). FMO is the best procedure todetermine the background fluorescence in the channel ofinterest, although does not provide information about thedegree of non-specific binding of the Ab. To this aim, thecombined use of FMO and IC might be useful to betterdetermine the boundary between the +ve and –ve popu-lation. However, the use of manufacturer-recommendedvolumes of IC often results in an overestimation of thebackground fluorescence. Here, we address this problemshowing a method to titer the IC for multicolor experi-ments. This method was used to determine the numberof CD38+ve T-cells in 3 healthy donors (HD). First, theserially diluted IC phycoerythrin (PE)-conjugated and thetitered CD38 PE-conjugated Abs were incubated with afixed number of antibody-capture beads. IC titer wasinterpolated using IC dilution curve, from the point inwhich CD38 and IC had the same value of MedianIntensity of Fluorescence. Results show that the numbersof CD4CD38 and CD8CD38+ve cells obtained using thetitered IC are intermediate (median CD4CD38 for the 3HD: 230cells/ul; median CD8CD38: 79cells/ul) betweenthose obtained with the FMO control only (medianCD4CD38: 258cells/ul; median CD8CD38: 100cells/ul)and with the untitered IC (median CD4CD38: 186cells/ul;median CD8CD38: 34cells/ul). This method could allow amore confident definition of the boundary between –veand +ve cell population, in case of antigens following acontinuous pattern of expression. In particular, it could beuseful in the analysis of dimly expressed antigens.

Oncology

immunofluorescence assays, but molecular fluorophoreonly used in bioconjugations are not particularly photo-stable and the intensity of the emitted light may be pHdependent or quenched by external agents.Dye-doped silica nanoparticles (NPs) help to overcomethese limitations, because the matrix acts as a protectiveshell against interfering species; moreover the molecularrecognition event is signaled from all the dyes included inthe nanoparticles. Silica also offers many advantages,being transparent to UV-Vis light, biocompatible, nontoxic, inexpensive and extreme versatility.Silica nanoparticles are fabricated and subsequently con-jugated to antibodies.The manufacture of our bioconjugates involves a prelim-inary activation of NPs with a specific cross-linker fol-lowed by the antibody modification using a reducingagent and the conjugation of the two species. The conju-gation mixture is purified by Size ExclusionChromatography and by Affinity Chromatography toachieve the maximum grade of purity. Our result show agreat level of purity (about 96% of purity) because of thegood separation between every single component of ourconjugation bulk that we can easily isolate through thecombination of two consecutive purifications.Our dates suggests that the bioconjugated silica nanopar-ticles are optimal reagent for immunfluorescense assay asflow cytometry and CellSearch apparatus, a system to iso-late, collect and quantify rare cells. The advantages of theirapplication are a reduced background, better discrimina-tion between different cellular population, high fluores-cence emission, compatibility with standard staining proce-dure and analysis, high stability in the time.

HIGHER LEVEL OF TRANSFORMING GROWTH FAC-TOR-ββ1 IN TUMOR-DRAINING PULMONARY VEINTHAN PERIPHERAL BLOOD IN PATIENTS WITH NONSMALL CELL LUNG CANCER: A POSSIBLE MARKEROF TUMOR PROGRESSIONCamerlingo R., Sessa G., Irollo E., Miceli R. and Pirozzi G.O.U. Cancer Stem Cell – Research Department I.N.T.-IRCCS “G.Pascale” of Naples, Italyr.camerlingo@istitutotumori.na.it

Objectives: Transforming Growth Factor-β1 (TGF-β1) is amultifunctional factor that regulates cellular differentia-tion, proliferation, motility and is considered one of themain Epithelial-Mesenchimal Transition (EMT) inductors.Its has been found to be overexpressed locally in manytumors and it plays an important role in tumor transforma-tion and progression. In order to investigate the possiblerole of TGF-β1 in patients with Non Small Cell LungCancer (NSCLC) as a marker of metastatic disease, weevaluated its level in plasma from the peripheral blood(PB) compared to that from draining pulmonary vein(RP), the blood vessel closer to the tumor site.Methods: We analyzed the TGF-β1 plasma levels of 61patients with NSCLC, enrolled from little more than ayear. The TGF-�1 plasmatic concentration, expressed inpg/mL, was assayed by FlowCytomix-Human Basic Kitand the results obtained were analyzed by theFlowCytomix Pro program, all provided by eBioscience.Results: In 25 of the 61 patients (41%) the TGF-β1 levelfrom RP, wich ranges from 5,8ug/mL to 53,8ug/mL with amean value of 23,4ug/mL, was higher than in the PB,which ranges from 5,2ug/mL to 37,1ug/mL with a meanvalue of 14,9ug/mL. In one of the 25 cases it wasobserved an increase in the concentration of TGF-β1 ofmore than 15 times of the RP compared to PB. No corre-lation with histopathological and clinical data was found.

Discussion: Metastasis is responsible for more than 90%of cancer associated mortality. For distant metastasis,primary tumor cells must invade, disseminate throughblood vessels, seed at the distant site, and colonize tomacrometastases. From our results no correlation withclinical data was found because all patients were enrolledfrom little more than a year but high levels of TGF-β1 rightin the vessel proximal to the tumor (41% of patients ana-lyzed) could be indicative of metastatic disease.

STUDY ON EXPRESSION OF HSA-MIR 17-3P INHUMAN GLIOMA T98G CELLS TREATED WITH THEANTI-OXIDANT DRUG GALLIC ACIDCapelli E.1, Curti V.1, Paolini A.2,3, Pasi F.2, Mazzini G.2,4,Nano R.21Department of Sciences and Technologies for theEnvironment, University of Pavia, Pavia, Italy; 2Departmentof Biology and Biotecnologies, “Lazzaro Spallanzani”,University of Pavia, Pavia, Italy; 3Departments of MedicalPhysics, IRCCS Policlinico S. Matteo Foundation, Pavia,Italy; 4IGM-CNR, 27100, Pavia, Italyenrica.capelli@unipv.it

miRNAs are small (22-25nt) non coding RNA moleculesthat are emerging as important regulators of biologicalprocesses. These molecules play a major role in post-transcriptional gene silencing and alterations in miRNAsexpression are described in many different human dis-eases, including cancer. Several micro RNAs have beenidentified with potential importance in glioma biology andthis may identify potential therapeutic microRNAs target-ing functions relevant to both tumor biology and neuro-genesis. It has been shown that gallic acid, one of theemerging candidate molecules with anti-cancer activity,contrasts tumor proliferation by suppressing angiogene-sis, invasiveness and cell viability of U87 and U251glioma cells in vitro. This drug is a basic component unitof tannic acid widely distributed in the plant kingdom withantiviral and antioxidant activity. In the present study, westudied the expression of hsa-miR-17-3p, a novel microRNA that has been demonstrated to suppress tumori-genicity of prostate cancer and that inhibits mitochondrialantioxidant enzymes in human glioma T98G cells, withthe aim to demonstrate a relationship between the anti-tumor effects of gallic acid and the down regulation ofantioxidant activities. Our results demonstrated that gal-lic acid induces a progressive increase in cell death andin the expression levels of hsa-miR-17-3p with theincreasing concentration of the drug (dose range: 1-100ng/ml). On the contrary mitotic index is progressively sig-nificantly reduced. Considering these our preliminaryresults and that hsa-miR-17-3p is involved in manyimportant signalling pathways (i.e. MAPK, neurotrophin,mTOR, Insulin) the use of gallic acid or its derivatives inthe treatment of glioblastoma could represent the begin-ning of new promising therapeutic strategies.

ANTIANGIOGENIC POTENTIAL OF THE TELOMERE-TARGETING AGENT RHPS4: INSIGHT INTO THEMECHANISM OF ACTION OF A NEW CLASS OF ANTI-CANCER MOLECULESD’Angelo C.1, Zizza P.1, Salvati E.1, Cingolani C.1,Porru M.1, Stoppacciaro A.2, Leonetti C.1, Biroccio A.1. 1Experimental Chemotherapy Laboratory, Regina ElenaCancer Institute. Rome, Italy. 2Department of ExperimentalMedicine, La Sapienza University. Rome, Italy.dangelo@ifo.it

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Angiogenesis, the process of new capillary formationfrom pre-existing vessels, plays an essential role in theembryonic and postnatal development, in the remodellingof various organ systems, and in several pathologies,including cancer. In the last context, it has long beenappreciated that tumour growth and progression aredependent on angiogenesis and, on the basis of theseresults, a growing number of anticancer treatments –both conventional and novel targeted therapies – haveshown to directly or indirectly suppress the neovascula-ture in established tumours.Here, we have specifically investigated the effect ontumour angiogenesis of the pentacyclic acridine RHPS4, atelomere-targeting agent having antitumour activitythrough its ability to induce DNA damage. Our data clearlydemonstrate that RHPS4 treatment induces a robust inhi-bition of cell migration, invasion and vessel formation.Moreover, the experiments performed on both humanumbilical vein endothelial cells (HUVEC) and mousepolioma middle T transformed endothelial cells (H5V),demonstrate that RHPS4 acts at the level of monomericGTP-ases of the Rho family (i.e. RhoA and Rac1) by neg-atively regulating the reorganization of actin cystoskeleton.Altogether these results reveal a novel, and so far unde-scribed, antiangiogenic effect of RHPS4 and suggest apeculiar role of this potential anticancer drug againstthose malignancies requiring neovasculaure for growthand spread.

THE METASTATIC POTENTIAL OF TUMOR CELLSCAN BE REVEALED BY 3D CULTURE ON A SILICONOPTICAL MICROCHIPDanova M.,1 Erba E.,4 Mazzini G.,2,3 Scovassi A.I.,2Aredia F.,2 Panini N.,4 Torchio M.,1 Barillaro G.,5

Surdo S.,,5 Carpignano F.,6 Silva G.6 and Merlo S.6

1Medicina Interna e Oncologia Medica, AziendaOspedaliera di PAVIA, 2Istituto di Genetica MolecolareIGM-C.N.R; 3Dipartimento di Biologia e Biotecnologie ‘‘L.Spallanzani’’, Università di PAVIA, 4IRCCS-Istituto diRicerche Farmacologiche “Mario Negri”,Unità diCitometria, MILANO, 5Dipartimento di Ingegneriadell’Informazione, Università di PISA, 6Dipartimento diIngegneria Industriale e dell’Informazione, Università diPAVIA - Italymazzini@igm.cnr.it

Background. The metastatic process includes a series ofsteps in which cancer cells migrate or flow through differ-ent microenvironments (ME). The ability to successfullyoriginate a secondary tumor is partially dependent on thephysical interactions and mechanical forces betweencancer cells and the ME. In this process, in particular,cells must undergo large elastic deformations. A betterunderstanding of the role of biophysical and mechanicalproperties of cells will provide new and important insightsinto the progression of cancer and could provide thebasis for new diagnostic/prognostic approaches. Weinvestigated the cell membrane plasticity as an indicatorable to forecast the tumor cell behavior with regard tomobility and/or migration. Both these actions are stronglyrelated to the cell metastatic potential (MP). Methods. Silicon micromachined structures (SmS) wereexploited as 3D microincubators able to host tumor celllines with different level of MP. The 3D microstructuresselected for this research consist in periodic arrays of sil-icon walls, with thickness of a few µm, separated byempty gaps (width of 5 µm), and high vertical aspect-ratio(height up to 100 µm), fabricated by electrochemicalmicromachining of silicon dice. After incubation (24-

48hrs) SmS samples were washed in PBS and fixedovernight in cold 70% ethanol and later stained withFITC/PI for fluorescence microscopy. Cell culture experi-ments were performed in triplicate on 4 different estab-lished cell lines, two of them with high MP (MDA-MB-231,RPMI-7951) and 2 with low MP (MCF-7, CAPAN-1). Cellsfrom the K562 line were used as the negative control.Results/Conclusions. Fluorescence microscopy of SmSpopulated by cells clearly evidenced the following behav-ior: a) K562 cells were found only on the surface of SmS;b) low MP cells are largely unable to grow on the SmSsurface and just a few cells can survive on the top of thewalls (their nuclei appear to have round shapes typical ofcells growing on flat surfaces); c) high MP cells can growon the SmS thanks to their ability to colonize the narrowempty space between silicon walls (red stretched nucleiall aligned along the wall direction clearly prove that thesecells are deeply inside the extremely narrow gaps of theSmS). The less stiff (more “plastic”) cytoskele-ton/membrane of this second type of cells (more metasta-tic) corresponds to their bio-mechanical ability triggeringtheir “clinical aggressivity” also exhibited on SmS: thesestructures thus become an interesting candidate to beimplemented in a lab-on-chip for clinical testing.Work partially supported by Fondazione Cariplo, grantno. 2011-0308.

THE MET ONCOGENE IS A FUNCTIONAL MARKEROF A GLIOBLASTOMA STEM CELL SUBTYPEDe Bacco F.1, Casanova E.1, Medico E.1, Pellegatta S.2,Orzan F.1, Albano R.1, D’Ambrosio A.1, Pollo B.2,Comoglio P.M.1, Finocchiaro G.2, Boccaccio C.11Institute for Cancer Research @ Candiolo, University ofTurin Medical School, Candiolo, Italy; 2IstitutoNeurologico C. Besta, Milan, Italycarla.boccaccio@ircc.it

The existence of treatment-resistant cancer stem cellscontributes to the aggressive phenotype of glioblastoma.However, the molecular mechanisms that sustain stemcell proliferation in these tumors are largely unknown. Wereport that expression of the MET oncogene, encodingthe HGF Receptor, identifies a subset of glioblastoma-derived neurospheres (MET-pos-NS), characterized by agene expression profile classified as mesenchymal orproneural. Conversely, neurospheres that do not expressMET (MET-neg-NS) usually harbor an amplified EGFRgene and display a gene expression profile defined asclassical. We show that MET-pos-NS are hierarchicallyorganized, and include a cell subpopulation expressinghigh levels of MET (MET-high) at the top, and a cell sub-population that conversely express negligible levels ofMET (MET-low), at the base. This conclusion is support-ed by data showing that, upon flow cytometric isolationfrom neurospheres, (i) MET-high, but not MET-low, cellsretain long-term propagation and multi-potential differen-tiation; (ii) MET-high, but not MET-low, cells can regener-ate a mixed MET-high/MET-low cell population; (iii) MET-high cells display increased tumorigenic ability as com-pared to MET-low cells, and are the only that form tumorscontaining both MET-high and MET-low cells. In MET-high cells, we also find that HGF sustains the stem cellphenotype (expression of self-renewal markers, clono-genicity and invasion) in vitro. Together, these findingssuggest that MET is a functional marker of glioblastomastem cells, and a promising target for identification andtherapy of a subset of glioblastomas.

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CANCER STEM CELLS IN HEAD AND NECKTUMORS: EVIDENCE FOR METASTATIC SPREADAND TREATMENT RESISTANCEDesiderio V.1, Tirino V.1, Paino F.1, La Noce M.1, andPapaccio G.11Dipartimento di Medicina Sperimentale, SecondaUniversità di Napoli, Italyvincenzo.desiderio@unina2.it

The major challenge in the management of patients withoral squamous cell carcinomas (OSCC) is the develop-ment of resistance to therapy leading to disseminateddisease. Since cancer stem cells (CSC) have emergedas important players in OSCC metastasis, our objectiveswere to explore the implications of CSC in OSCC tumorprogression, invasion and response to conventional ther-apies. Methods: A panel of well-characterized cell linesoriginated from the most common sites in the head andneck area was used. Cells were cultured as spheres orunder normal adherent conditions and analyzed forCD44, ALDH, CD24, CD29, CD56 by flow cytometry,PCR arrays for genes related to stemness, metastasisand EMT. We also investigated sLeX expression, knownto play a key-role in many cancers metastasis by promot-ing tumor cells binding to endothelial E-selectin. We ana-lyzed the tumorigenic potential of OSCC cells by invasionassays and in vivo OSCC experimental models compar-atively to CSC cells. Resistance to cisplatin and radiationwas assessed by annexin V/PI assay and colony formingassay. Results: The highest levels of sLeX expressionwere found in cell lines originated from oral cavity (9%-47%) compared to other head and neck locations (0.1%-7%). Cells grown as spheres were 95-100% positive forsLeX compared to 10-40% of adherent counterpart.Although sLeX+ and sLeX- cells were both able to formspheres, sLeX+ spheres were predominant and larger.Flow cytometry and PCR arrays indicated that thespheres were highly enriched in CSC and metastaticmarkers. Consistently, the spheres showed increasedinvasive and tumorigenic potential, and resistance to con-ventional chemotherapy and radiations. Conclusion: thisstudy is the first to unveil a novel link between sLeXexpression, stem cell formation and metastatic spread inOSCC, and provides supportive evidence for CSC resist-ance to treatment. Understanding the mechanisms oftumor invasion and metastasis will improve patient out-come and survival.

MULTIPLEX FLOW CYTOMETRY IDENTIFICATION OFSURFACE SPECIFICITIES OF PRIMARY ANDMETASTATIC COLON CANCERSGemei M.1, Galizia G.2, Mirabelli P.3, Di Noto R.1,4,Del Vecchio L.1,4.1CEINGE – Advanced Biotechnologies, Naples, Italy; 2Divisionof Surgical Oncology - “F. Magrassi - A. Lanzara” Department ofClinical and Experimental Medicine and Surgery - SecondUniversity of Naples School of Medicine, Naples, Italy;3Fondazione SDN-IRCSS, Naples, Italy; 4Department ofMolecular Medicine and Medical Biotechnologies (DMMBM),University of Naples “Federico II” Naples, Italymaricagemei@gmail.com

Identification of tumor associate antigens (TAA) is funda-mental for prognostication, early detection and targeting ofcancer cells. Cancer associate surface antigens can be alsoimportant target for molecules and drugs systemically deliv-ered, functioning through the block of signaling pathways orantibody-dependent cell- and complement-mediated cyto-toxicity. Flow cytometry allows the analysis of protein

expression in situ and at single cell level. We analyzed, bymultiplex flow cytometry, the expression of 27 surface anti-gens comparing their expression in normal colon, colon can-cers and hepatic metastases. We determined a specific sur-face signature of colon cancer cells identifying putative func-tionally important proteins whose expression was signifi-cantly higher in primary tumors from metastatic patients ascompared to non metastatic ones, as well as in metastasissamples as compared to primary tumors. We first comparedthe 27 antigens expression in normal colon and in colon can-cer tissue. The expression level of 16 antigens (CD133,CD24, CD29, CD44, CD47, CD49b, CD49f, CD54, CD55,CD59, CD66b, CD66c, CD81, CD164, CD166, CD227) wassignificantly (p<0.05) enhanced in cancer samples respectto normal ones. We also observed an expansion of CD133,CD24, CD55, CD66c, CD221, CD227 expressing cell popu-lations in tumor tissues respect to normal colon. Additionally,hepatic metastases had a significantly higher expression ofCD24, CD26, CD66c and CD151(p=0.0099, p=0.0180,p=0.0119, p=0.0395 respectively). We also analyzed theexpression of the 27 markers on CD133+ and CD133- sub-sets of cancer samples to evaluate differences between thestem-like compartment and the more differentiated one. Theexpression level of 15 antigens was significantly (p<0.05)enhanced, in primary tumors, in CD133+ cells respect toCD133- ones. The stem compartment from hepatic metas-tases revealed significantly (p=0.0062) higher expression ofthe integrin CD29. Finally, one of the differentially expressedantigens: CD26/DPPIV, was found on circulating cancercells from peripheral blood of colorectal cancer patients. Thepresence of the CD26+ circulating cells strongly correlatedwith cancer recurrence for which it was a prognostic factor inmultivariate analysis.

MYCN GENE EXPRESSION IS REQUIRED FOR THEONSET OF DIFFERENTIATING PROGRAM IN CELLSOF NEURAL ORIGINGuglielmi L., Cinnella C., Maresca G., Felsani A. andD’Agnano I.CNR-Institute of Cell Biology and Neurobiology, Rome, Italyloredana.guglielmi@ibcn.cnr.it

The MYC family gene, MYCN, plays a role during the firststeps of human development. It is widely accepted thatMYCN decreases during differentiation in cells of neuralorigin. We show that MYCN expression increases inembryonic cortical neural precursor cells after few daysfrom differentiation induction. We used human neuroblas-toma cell lines as model to investigate the role of MYCNin the earlier stages of neuronal differentiation. In LAN-5neuroblastoma cells, MYCN is up-regulated after 2 daysof retinoic acid treatment before its expected down-regu-lation. A positive modulation of some differentiation mark-ers, such as CDK5, ENO2, SLC18A3, MAPT/TAU,GAP43 and ChAT, is associated with the increase ofMYCN expression. Likewise, MYCN silencing inhibits dif-ferentiation in LAN-5 MYCN knock-down (KD) clones,leading to a negative modulation of the aforementioneddeveloping markers. In addition, the LAN-5 MYCN KDclones show a loss of neurofilaments formation and adepolarization of the plasma membrane. Furthermore,overexpression of MYCN gene in the poorly-differentiat-ing neuroblastoma cell line SK-N-AS restores the expres-sion of differentiation-related genes as well as neuritisoutgrowth. NF200 immunostaining reveals an increasedability to form mature neurofilaments in SK-N-AS MYCN+clones, after retinoic acid treatment. MicroRNA Assayanalysis revealed that some specific miRNAs (miR9,miR20a and miR92a) are conversely modulated during

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try. A549 cell fractions obtained were treated with TGFβ1for 48 hours. After incubation, were analyzed by immuno-fluorescence, RT-PCR and Western Blot. Migration,Wound-healing were performed to assess the migratorycapacity. Soft agar assay was performed to evaluatetumorigenicity.Results: TGFβ1 induced EMT in all fractions of A549 sort-ed. It increases the percentage of cells migrating except forCD133- fraction. The migration of SP- cell fraction isstronger than of its untreated corresponding cells. Althoughthis, CD133+ cell fraction responded more effectively to theTGFβ1 treatment. For stemness, TGFβ1 induced anincrease of OCT4 in all treated fractions except for CD133-

. Wound size reveals that TGF-β1 enhances motility in allcell fraction except for CD133- and SP- cells. Assessmentof growth kinetics reveals major colony-forming efficiencyin CD133+ A549 cells. For CD133- cells no change incolony number was observable.Conclusion: The results obtained suggest that TGFβ1 high-lights different stemness subpopulations with different char-acteristics: i) a stationary CSCs subpopulation identified asSP+ cell subset and ii) a migratory CSCs subpopulationidentified as CD133+ cell subset. Each population seems tobe involved in different biological mechanisms such as stem-ness maintenance, tumorigenicity, invasion and migration.Noteworthy and interestingly, also SP- showed strong migra-tory capacity indicating TGFβ1 could induce cell reprogramon non-CSCs by EMT leading to coexistion by switchingbetween the two phenotypic states.

ABSENCE OF LAMIN A/C AS POSSIBLE MARKER OFCANCER STEM CELLS IN NEUROBLASTOMANardella M.1, Guglielmi L.1, Amendola D.2, Maresca G.1,Sessa G.3, Valentini A.4, Pirozzi G.3, Bucci B.2, Felsani A.1and D’Agnano I.11CNR - Institute of Cellular Biology and Neurobiology, Rome;2Center for Scientific Research S. Pietro FatebenefratelliHospital, Rome; 3Research Dep., INT Pascale, Naples;4Departments of Laboratory Medicine and of Internal Medicine,PTV University of Tor Vergata, Rome, Italymarta.nardella@ibcn.cnr.it

A-type lamins, major constituents of the nuclear lamina,are absent in most types of stem cells. In a previouspaper we have shown that silencing of lamin A/C blockeddifferentiation in neuroblastoma cells. Neuroblastomamay be considered as a heterogeneous population ofcells in which only a minor population capable of tumorinitiation: the cancer stem cells. Our aim is to study therole of A-type lamins in the development of neuroblas-toma as stem cells disease. We used the SH-SY5Y neu-roblastoma cell line. Silencing of lamin A/C causes anincrease of the expression of the stemness markers suchas CD133/Prom-1. CD133+ cells were isolated fromLMNA-KD cells using magnetic cell sorting technology.The CD133(+)-enriched cell population showed anincreased expression of the stemness markers. Theacquisition of a stem cell-like phenotype is also support-ed by the presence of a “side population”. Consistently,the LMNA-KD cells expanded as non-adherent second-ary sphere-like cellular aggregates in serum-free mediacontaining EGF and FGF2. When tumour spheres werecultured without EGF and FGF-2 in the presence of 10%serum floating cells attached to the plastic as monolayer.This cell line retains the same stem cell phenotype. In theLMNA-KD cells, miRNAs expression profiling evidencedthe modulation of a different number of miRNAs involvedin differentiation, such as miR-124 and miR-101. Downregulation of miR-101 is consistent with the increased

the silencing and overexpression of MYCN gene in thetwo different cell lines, indicating a possible role of thesenon-coding RNAs in driving the onset of differentiatingprograms.

A POSSIBLE ROLE OF LAMIN A/C IN ChAT-ACTIVATEDDIFFERENTIATION MOLECULAR MACHINERYGuglielmi L.1, Nardella M.1, Arisi I.2, Storti AE.2, D’Onofrio M.2,Cacci E.3, Biagioni S.3, Tocco MG.3, D’Agnano I.1 andFelsani A.11CNR-Institute of Cellular Biology and Neurobiology;2European Brain Research Institute; 3Dep. of Biology andBiotechnology «Charles Darwin», Sapienza University;Rome; Italyloredana.guglielmi@ibcn.cnr.it

The mouse neuroblastoma N18TG2 clone is defective forbiosynthetic neurotransmitter enzymes; thus appearingalways as an immature neuronal clone. The forced expres-sion of ChAT in these cells results in the synthesis ofendogenous ACh and in their higher ability to express neu-rospecific features and markers. We investigated more indetail the wide genome expression profiling differencesbetween the N18TG2 clone and its ChAT-transfectedderived clone (2/4). The developmental program switchingon in the 2/4 clone was confirmed by the increasingexpression of several differentiation-related genes (CDK5,CEND1, Syn1, Syn2, Nav1, Tubb3) and by the decrease oftypical cell cycle-related genes expression, such asCyclins and Cdks. We also observed an increase of LMNAgene expression in 2/4 clone; interestingly, several studiesdemonstrated Lamin A/C shows a differentiation-relatedaccumulation. In fact, we stimulated the differentiationprocess with retinoic acid and we observed an increasingexpression of Lamin A/C protein in both clones during dif-ferentiation stimuli. We performed agonist/antagonistreceptor study to establish if LMNA expression was strictlyrelated to AChR activation: carbachol treatment (AChRagonist) led to increasing LMNA as well as neuronal differ-entiation markers expression in N18 cells; nevertheless,treating 2/4 clone with atropine or mecamylamine (AChRantagonist) caused a diminution in LMNA mRNA levels anda concomitant decrease of differentiation related genes.Our hypothesis is that Lamin A/C might be involved in theChAT-dependent molecular differentiation pathway, hencewe are trying to corroborate this putative role by silencingLMNA gene in 2/4 clone.

TGFββ1 HIGHLIGHTS SEVERAL SUBPOPULATIONSWITH DIFFERENT CHARACTERISTICS OF STEMCELL THAT LEAD TO METASTASISIrollo E.1, Tirino V.2, Camerlingo R.1, Sessa G.1, Miceli R.1,Rocco G.3 and Pirozzi G1.1Department of Experimental Oncology, National CancerInstitute, Naples, Italy. 2Department of ExperimentalMedicine, Second University of Naples, Naples, Italy.3Department of Thoracic Surgery and Oncology, NationalCancer Institute, Naples, Italy.e.irollo@istitutotumori.na.it

Objectives: Metastasis is the leading cause of death bycancer. Non-small-cell lung cancer represents nearly 85%of primary malignant lung tumours. The epithelial-to-mes-enchymal transition (EMT) plays a key role in metastasis.TGFβ1 is the major inductor of EMT. Aim of this study wasto investigate the role of TGFβ1 on cancer stem cells(CSCs) and non-CSCs starting from A549 lung cell line.Methods: A549 cell line was sorted for CD133 surfaceantigen and Side Population (SP) profile by flow cytome-

levels of MYCN in LMNA-KD cells. We hypothesize thatA-type lamins are necessary to maintain low levels ofMYCN by regulating miR-101 expression.

EXCITOTOXIC DEATH INDUCED BY GLUTAMATE INRAT CEREBELLAR GRANULE CELLS IS INHIBITEDBY SILENGING LAMIN A/CNardella M.1, Ciotti MT.1, Storti AE.2, Brandi R.2, Arisi I.2,D’Onofrio M.2, Florenzano F.3, Felsani A.1 and D’Agnano I.11CNR - Institute of Cell Biology and Neurobiology, Rome;2European Brain Research Institute EBRI - NeurogenomicsIIT Unit, Rome, 3European Brain Research Institute EBRI -Confocal Microscopy Unit, Rome, Italymarta.nardella@ibcn.cnr.it

The cerebellum granule cells (GCs) are the most numerousneurons of the entire central nervous system and lie withinthe internal granular layer, located below a monolayer ofPurkinje cells. The maturation and differentiation of GCs ispromoted by excitatory amino acids receptors activation,such as glutamate. However, an excessive activation maycompromise the cell survival, this phenomenon (excitotoxiceffect) being involved in many neurodegenerative diseases.The GCs provide an excellent experimental model formolecular and cell biological studies of neuronal develop-ment and neurodegenerative diseases, because their devel-opment in vitro mimics very well that in vivo, thus preservingthe same mechanisms of glutamatergic fibers formationoccuring during development. Primary cultures of post mitot-ic cerebellar GCs represent a homogeneous cellular systemshowing a single neuronal population since glia expansion isprevented by inhibiting mitosis. The primary cultures of GCshave a short life, in fact as their maturation progressesbecome gradually more and more vulnerable to the effect ofglutamate, and after eight days of culture they reach their fullmaturity and die by necrosis. Our aim is to study whether A-type lamins, know to play a crucial role in the differentiation,can influence the granule neurons maturation. By immuno-histochemistry, we have seen that Lamin A/C expressionincreases during the development of GCs in vivo. We mon-itored neurons maturation in vitro studying the cell responseto stimulation by glutamate. Silencing of LMNA gene inhibit-ed glutamate-mediated spontaneous neuronal death. Thussuggests that the LMNA knock down in GCs exhibits a sortof glutamate-resistant phenotype, which has been charac-terized by performing genome wide profiling.

EFFECTS OF GALLIC ACID ON VIABILITY, PROLIFERA-TION AND INVASION IN HUMAN GLIOMA T98G CELLSPaolini A.1,3, Pasi F.1, Curti V.2, Mazzini G.1,4, Capelli E.2,Nano R.11Department of Biology and Biotecnologies, “LazzaroSpallanzani,” University of Pavia, Pavia, Italy; 2Departmentof Sciences and Technologies for the Environment,University of Pavia, Pavia, Italy; 3Departments of MedicalPhysics, IRCCS Policlinico S. Matteo Foundation, Pavia,Italy; 4IGM-CNR, 27100, Pavia, Italyalessandro.paolini01@ateneopv.it

Glioblastoma multiforme (GBM) is the most common andmost malignant primary brain tumor in adulthood. Despitetreatment efforts including new technological advances inneurosurgery, radiation therapy, and clinical trials withnovel therapeutic agents, the tumor recurrence is veryhigh. Therefore, it is of critical importance that new effec-tive treatments for glioblastoma must be developed.Gallic acid, an organic acid found both free and as part of, has been shown to inhibit carcinogenesis in animalmodels and in vitro cancerous cell lines. Previous studies

have demonstrated that this molecule inhibits cell viabili-ty of U87 and U251 glioma cells in a dose-dependentmanner. As such, the aim of this study is to evaluate theeffects of gallic acid, in T98G human glioma cell line, ,which it is known to have a high invasion ability and to bechemoresistant. We have performed viability, proliferationand invasion tests using five different concentrations. Ourdata demonstrated that gallic acid reduces glioma cellsproliferation decreasing the mitotic process, but only highconcentrations (100µg/ml) of gallic acid are able toinduce cell death. We assume that T98G cells are moreresistant than other glioma cell lines to gallic acid treat-ment because we obtained data similar to the literatureonly after three time higher concentrations of the drug. Inconclusion gallic acid may be a valuable candidate fortreatment of brain tumour in association with other morecytotoxic treatments.

ANALYSIS OF ANTITUMOR ACTIVITY OF TRABECTE-DIN IN MYELODYSPLASTIC/MYELOPROLIFERATIVENEOPLASMSRomano M.1, Galli A.2, Aronica A.3, Picone C.2, Panini N.1,Paracchini L.1, Tancredi R.4, Marchini S.1, Rosti V.3,Della Porta M.2, D’Incalci M.1 and Erba E.11IRCCS-Istituto di Ricerche Farmacologiche Mario Negri,Milano; 2Department of Hematology Oncology, Fonda zioneIRCCS Policlinico San Matteo,University of Pavia; 3Center forthe Study of Myelofibrosis, Research Laboratory ofBiotechnology, Fondazione IRCCS Policlinico San Matteo,Pavia. 4Unità di Oncologia, Lab. Oncologia Sperimentale eFarmacogenomica, IRCCS Fondazione Maugeri, Pavia Italyeugenio.erba@marionegri.it

Juvenile myelomonocytic leukemia (JMML) and chronicmyelomonocytic leukemia (CMML) are myelodyspla stic/mye loproliferative neoplasms (MDS/MPN) of childhood andelderly patients, respectively. Current treatment options forJMML and CMML are highly unsatisfactory and the onlycurative therapyis allogenic BM transplantation. There is cle-arly a need to identify new effective treatment strategies forthese patients. Trabectedin, a new DNA binder of marine ori-gin was recently found to cause selective depletion of mono-cytes in blood and spleens of tumor-bearing mice, to activa-te caspase-8-dependent apoptosis, and TRAIL receptorsexpression. The aim of this study was to characterize the invitro cytotoxic effect, the cell cycle phase perturbations, theexpression of TRAIL receptors and the mechanism of celldeath induced by trabectedin on biphenotypic B-myelomo-nocytic leukemia MV4-11 cell line and on the malignant mye-lomonocityc cell compartment of patients suffering fromCMML or JMML.As observed on MV4-11 cell line, in the first 20 cases wefound that malignant monocytes from both JMML andCMML are highly susceptible in vitro to trabectedin com-pared to azacytidine and cytosine arabinoside (ara-c).Trabectedin, at nanomolar concentrations, was able toinduce strong apoptosis, evaluated by flow cytometricannexin-V/PI assay, both on MV4-11 cells and on mali-gnant monocytes from patients after 24, 48 and 72h tre-atment. On MV4-11 cells, apoptosis was found to becaspase-8-dependent. Trabectedin, on MV4-11 cells,induced the expression of TRAIL receptors, evaluated byflow cytometry, after 24 h treatment at the concentrationof 0.5 nM, while in malignant monocytes from JMML andCMML did not. In addition, trabectedin inhibited in a dosedependent manner the clonogenic growth of myelomono-cytic progenitors of CMML patients and the spontaneousgrowth of circulating CFU-GM of JMML patients, respec-tively. Molecular studies on malignant monocytes from

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JMML and CMML treated with trabectedin are in progressto identify molecular pattern associated to clinical outco-me. All togehther, our data suggest that Trabectedincould be a candidate for a new effective treatment inMDS/MPN, and in particular in JMML and CMML.

GENE EXPRESSION PROFILING OF SPHERES DERIVEDFROM HUMAN PRIMARY CHONDROSARCOMATirino V.1, Desiderio V.1, Paino F.1, Nebbioso A.2, Altucci L.2,Pirozzi G.3, La Noce M.1, De Rosa A.4, and Papaccio G.11Dipartimento di Medicina Sperimentale, Seconda Universitàdi Napoli, Italy. 2Dipartimento di Biofisica, Biochimica ePatologia Generale, Seconda Università di Napoli, Italy.3Dipartimento di Oncologia Sperimentale, Istituto Nazionaledei Tumori di Napoli, Italy. 4Dipartimento di Chirurgia eBiologia Orale, Seconda Università di Napoli, Italy virginia.tirino@unina2.it

Background and Purpose: Solid tumours are made of cellsubsets defined cancer stem cells with stemness poten-tials, aggressiveness and increasing expectancy of recur-rence. These cells are able to form spheres. Aim of thisstudy was to obtain chondrospheres from a primary chon-drosarcoma and to compare the molecular and cellularcharacteristics with corresponding adherent cells.Procedures: Primary chondrosarcoma cells were culturedboth in standard and sphere culture condition. On bothspheres and adherent cells, cytometric analyses forstemness and differentiation antigens including CD133,OCT4, Sox-2 and collagen type II were performed. Inaddition, cell cycle analyses and apoptosis assay wereperformed after cisplatin treatment performed for 48 h atdifferent and high concentrations. Gene expressing pro-file microarray was performed. Findings: Chondrospheres were formed already after 24hours of culture. They over-expressed CD133, OCT3/4, andSox2 markers and showed resistance to cis-platin treatment.Microarray analyses pointed out a huge gene expression dif-ference between chondrospheres and adherent cells. With acut-off threshold of 2-fold, 1405 genes were differentiallyexpressed of which 629 genes were over-expressed and 776under-expressed on chondrospheres. The most highly over-expressed genes were involved in multidrug resistance, cellcycle, apoptosis regulation, migration, motility and invasionregulations and stemness phenotype. Conclusions: This study highlights that chondrosarcomasare a source of cancer stem cells and chondrospheresmay constitute an excellent model to study stemness,tumoral heterogeneity and multi drug resistance to individ-uate candidate biomarkers for novel therapeutic targets.

SIDE POPULATION CHARACTERIZATION IN HEPA-TOCELLULAR CARCINOMA CELL LINES Trombetta E.,1 Colombo F.,1 Spagnuolo G.,1 Cetrangolo P.,1Prati D.,1,2 and Porretti L.,11Ffondazione IRCCS Ca’ Granda Ospedale MaggiorePoliclinico, Centro Interdipartimentale di Citometria,Milano, Italy - 2Oospedale A. Manzoni, Dipartimento diMedicina Trasfusionale ed Ematologia, Lecco, Italy.labcitofl@policlinico.mi.it

Side Population (SP) was initially identified as a populationof hematopoietic stem cells able to extrude from their cyto-plasm different drugs and the fluorescent dye Hoechst33342 through the activity of ATP-Binding Cassette (ABC)proteins. More recently, SP was also found in several tumorcell lines with stemness and chemoresistance properties.The aim of this study was to characterize SP in different HCCcell lines by means of the expression of ABCG2 and other ABC

pumps and stem cell genes such as NANOG and OCT4.Flow cytometry evaluation of SP and non-SP cells andABCG2 membrane expression were performed in HCC1,Hep3B, HepG2, HuH7, SNU475 and PLC/PRF/5 cell lines fol-lowing standard protocols. Real Time-PCR (RT-PCR) of ABCG2,ABCB1, ABCC1, MVP, NANOG and OCT4 was carried out onSP and non-SP cells, sorted by BD FACSAriaII for mRNA quan-tification. The t test was used to compare distibutions.Side population was detected only in HCC1, Hep3B,HepG2 and HuH7 cell lines (16%, 3.2%, 0.3% and 0.2%,respectively). Only 4% of SP-HCC1 expressed ABCG2on their membrane, in accordance with the results of RT-PCR confirming low levels of ABCG2 expression on SP-HCC1. Interestingly, SP cells of all the four cell linesexpressed higher levels of ABCB1 as compared to non-SP (p<0.011}). For the other ABC proteins tested, no sig-nificant differences were found between SP and non-SPcells. With regard to stemness genes, only SP-HepG2cells expressed higher levels of NANOG and OCT4 ascompared to non-SP cells1} (p<0.002).In conclusion, SP is detectable in several HCC cell lines andit is characterized by a high expression of ABCB1 ratherthan ABCG2. The upregulation of NANOG and OCT4 in SP-HepG2 cells supports the hypothesis that these cells havestemness properties. Further studies are needed to eluci-date the role of ABCB1 in HCC chemoresitance.

MONOCLONAL ANTIBODY-BASED CANCER THERAPYAND DEVELOPMENT OF TUMOR ANTIGENMIMOTOPES AS VACCINETuccillo F. M.1, Palmieri C.2, De Laurentiis A.2,Falcone C.2, Fiume G.2, Galandrini R.3, Capuano C.3,D’Armiento F.P.4, Arra C.5, Barbieri A.5, Dal Piaz F.6,Bonelli P.1, Buonaguro F.M. 1, Santoni A.7, Quinto I.2,Scala G.2

1Lab. Molecular Biology and Viral Oncogenesis, and 5AnimalFacility, Istituto Nazionale Tumori-Fond. Pascale-IRCCS,Napoli, Italy. 2Dept. Experimental and Clinical Medicine,University Magna Graecia, Catanzaro, Italy. 3Dept.Experimental Medicine, University La Sapienza, Roma, Italy.4Dept. Biomedical Sciences Advanced, University Federico II,Napoli, Italy. 6Dept. Pharmacy, University of Salerno, Fisciano(SA), Italy. 7Dept. Molecular Medicine-Istituto Pasteur-Fond.Cenci-Bolognetti, University La Sapienza, Roma, Italy.fmtuccillo@yahoo.it

The UN1 monoclonal antibody (mAb) was produced on thebasis of selective reactivity with human thymocytes. We identi-fied the antigen recognized by the UN1 mAb (UN1 Ag) as CD43glycoforms that were peculiarly expressed in fetal tissues andseveral cancer tissues, while unexpressed in normal tissuecounterparts. Based on the cancer-associated expression, theUN1 Ag was an attractive target for cancer immunotherapy.In this study, we describe that the parental administrationof the UN1 mAb delayed the tumor growth of UN1-posi-tive T-lymphoblastoid HPB-ALL cells upon mouseengraftment. Based on the screening of an M13 phage-displayed random peptide library (RPL) using the UN1mAb as bait, we selected UN1-like phagotopes thatspecifically bound to the UN1 mAb. We demonstratedthat the mice immunization with a selected UN1 phago-tope arose antibodies that specifically recognized theUN1 antigen in tumor tissues as well a synthetic peptidemimicking the UN1 epitope. Taken together these resultsindicated that the UN1 mAb: (1) was able to target in vivocancer-associated CD43 glycoforms and inhibit thegrowth of tumor xenograft in mice, and (2) was an usefultool to select UN1 mimotopes from RPLs as potentialcancer vaccine candidates

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Abinun M., 32 Adami C., 28, 38 Agabiti S., 28 Agrati C., 32 Albano R., 47 Allavena P.. 10Altieri S., 13Altucci L., 51 Alviano F., 31 Amadei B., 39 Amendola D., 45, 49 Amoroso FC., 39 Andreoli C., 18 Annunziata S., 28 Annunziato F., 36Anselmo A., 10 Aracri B., 18 Arcangeletti M., 16, 31, 8 Aredia F., 11, 42, 47 Arfini C., 41Arisi I., 49 ,50 Armstrong L., 41Aronica A., 50 Arosio D., 5Arra C., 51 Artoni P., 5Ascierto P.A., 38 Assennato S.M., 42 Attolico I., 26Avanzini M.A., 12, 39 Azzari C., 16, 19 Bacchetta R., 32 Bacigalupo A., 34Baffone W., 9 Baldanti F., 33 Baldi C., 33 Baldi E., 16, 19Balestra C., 15, 16Balia C., 14 Ballarini F., 13, 25 Barba C., 29 Barbieri A., 51 Barillaro G., 42, 47 Barni S., 12 Baroncelli GI., 36 Barone F., 18Bartolacci V., 31 Basile A., 23 Basso G., 28, 29 Battaglia A., 34 Battaglia F., 33, 34, 37Bencini S., 20, 26 Benedetto L., 38 Bensi T., 41 Bernocchi G., 12 Bernuzzi F., 7Betri E., 7 Betti L., 19 Biagioni S., 49 Biagiotti C., 26 Bianchini P., 20 Biondi A., 24, 29, 40Biroccio A., 46

Bloise A., 38 Bloise N., 12, 39, 43 Boccaccio C., 47Boccardo S., 35 Boero S., 35 Boi P., 16Bolognese A., 32 Bonaccorso P., 40 Bonanno G., 42Bonde JP., 18 Bonelli P., 51 Bonetti M.I., 26, 20 Bonifacino T., 42 Bonsi L., 31 Bordoni V., 32 Borgonovo G., 7Bortolussi S., 13 Bosi A., 20, 26Bottone M.G., 12Bozzano F., 31, 34 Brandi R., 50 Brogi D., 37 Brondi M., 5Bruno G., 21 Bruno R., 33Buccella F., 31 Bucci B., 45, 49 Bugarin C., 29, 40 Buldini B., 28 Buonaguro F.M., 51 Buracchi C., 10 Burgarella S., 40 Buzzonetti A., 34 Caccamo N., 24Cacci E., 49 Calabrese V., 45 Calarota SA., 33 Calcagno L., 41 Calcaterra F., 6Callegari B., 23Cambi M., 16, 19Camerlingo R., 44, 46, 49 Campana D., 5Campana R., 9 Cancedda L., 5Candiotto L., 23Cannizzo G., 24 Canonico B., 16, 31, 8 Canonico E., 23, 45 Cansolino L., 13Cant A. J., 32 Capalbo S. F. 21Caparello MC., 36 Capasso D., 13Capelli E., 46 , 50 Capobianchi M.R., 32 Capone M., 36, 38 Caporale R., 20, 20 Capuano C., 51 Caracò C., 38Carbone C., 38 Cardone E., 36 Carlomagno S., 37 Carpignano F., 42, 47 Carrega P., 35 Casasco A., 43Casanova E., 47Casotti R., 15, 16Castellano G., 13

Catacchio I., 23 Cataldi A., 13 Cattaneo A., 42 Catzola V., 34 Cavalli M., 30 Cavallo D., 17Cazzaniga G., 24, 29, 40 Cazzaniga V., 24Ceccarelli G., 12Cecconi M., 32 Celentano V., 13Celi A., 14 Cenderello G., 31 Cesana C., 29 Cesarini E., 8 Cetrangolo P., 51 Chiarenza A., 30 Chiericozzi M., 29 Chiesa A., 33 Cianchetti S., 14 Ciandrini E., 9Ciervo A., 17 Cima S., 33 Cimini E., 32 Cingolani C., 46 Cinnella C., 48 Ciotti MT., 50 Ciriello M., 26 Ciriello M.M., 41 Ciullini Mannurita S., 32 Clavio M., 25 Clerici A.M., 13Colarossi S., 21 Colombo F., 42 , 51 Colombo N., 25 Coluzzi S., 26 Comoglio P.M., 47 Comolli G., 33, 41 Consensi E., 26, 30 Consolini R., 36Conteduca G., 7, 33, 34Conticello C., 30 Contini P., 33 Coppo M., 41Corbella M., 33 Cornelio G., 22 Cosentino M., 35Cosmi L., 36 Costa R., 31 Curti V., 46, 50 Cusella De Angelis M.G., 12 Cutini I., 20 ,26 D’Agnano I., 7, 45, 48, 49, 50 D’Alterio C., 36 D’Ambrosio A., 47 D’Andrea L.D., 13D’Angelo C., 46 D’Arena G., 21, 22 D’Armiento F. P., 51 D’Auria F., 21 ,22, 26 D’Incalci M., 50D’Offizi G., 32 D’Onofrio M., 49, 50 Dal Bo V., 12 Dal Col J., 11 Dal Piaz F., 51 Damonte G., 33 Dan K.M., 30 Danova M., 41, 47

Authors’ index

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Dattola A., 22 De Bacco F., 47De Benedetto M.A., 23De Berardis B., 18De Colli M., 13 De Laurentiis A., 51 De Leo L., 37De Maria A., 31, 34 De Palma R., 34 De Pascali S.A., 12 De Rosa A., 51De Tullio G., 22 De Vita M., 34 Debora G., 18 Degan P., 18Del Vecchio L., 26, 48Del Zotto G., 31 Della Bella S., 6Della Porta M., 50 Della Porta M.G., 26, 30Delrio P., 36 Dentone C., 31, 34 Desiderio V., 48, 51 Di Biagio A., 31, 34 Di Cecilia M., 38 Di Gaetano R., 23 Di Gaetano S., 13di Giacomo V., 13Di Giuli C., 38 Di Giulio M., 13 Di Leo MG., 37Di Liberto D., 24 Di Maria V., 34 Di Marzo L., 23 Di Monta G., 38 Di Noto R., 48 Di Prisco S., 42 Di Raimondo F., 30Di Terlizzi S., 23, 44, 45 Diaspro A., 20 Dieli F., 24, 24 Dionigi P., 13Disarò S., 28 Divella C., 13Dolcetti R., 11Dolezel J., 5Dominici S., 9 Dominietto A., 34 Dongmo B., 35 Dutto I., 14Eleuteri P., 18 Erba E., 47, 50Facchinetti A., 45 Fadda M.R., 24 Fagnoni F.F., 39Fagnoni M., 43 Failli A., 36 Falcone C., 51 Falugi C., 20 Fanizzi F.P., 12 Farina A., 9, 17, 18 Fassina L., 43 Fattorossi A., 34Fazio G., 24, 40 Fedele R., 22 Felsani A., 48, 49, 50Fenoglio D., 7, 31 ,33, 34, 37 Ferrara I., 28 Ferrari C., 13

Ferrari N., 35 Ferrea G., 34 Ferrera F., 33, 34 Ferri U., 29 Ferrone S., 3 Festa L., 13Fiamenghi C., 7Figliuzzi M., 40 Filaci G., 7, 31, 33, 34, 37 Finocchiaro G., 47 Fiume G., 51 Florenzano F., 50 Focarete M.F., 39 Fontana R., 28 Fontana V., 35 Formentini A., 39 Forti G., 16, 19 Fossati G., 38 Fossati M., 34 Fraccaro P., 31 , 34 Francescato S., 28 Frassanito M. A., 23 Fresegna A. M., 17 Frugoni C., 42 Fugazza G., 25 Fumi M., 25 Gaipa G., 9, 24, 29, 40Galandrini R., 51 Galasso C., 15 Galizia G., 48 Galli A., 50 Galli L., 8 Gallorini M., 13 Gambardella C., 20 Gambineri E., 32Gasparetto V., 23 Gatti A. M., 20 Gatti A., 16Gaudenzi G., 25 Gavazzo P., 43Gelli A.M.G., 20 , 20 Gemei M., 48 Gennery A., 32 Gentilcore G., 38 Germano G. 10, Gervasi F., 27 , 27Gervasini C., 14Gesualdo L., 13 Geuna M., 26 Ghiggi C., 25 Ghio M., 33Giacomini M., 31 , 34 Gianfaldoni G., 20 , 26 Giannoni L., 25 Giansanti V., 11 Gigante M., 13 Gigli M., 39 Giorgi D., 9, 17Giribaldi F., 42 Girotto M., 26 Giudice V., 28 Giwercman A., 18 Gobbi M., 25 Grandaliano G., 13 Granzotto M., 37Grasso R., 25 Grimaldi A.M., 38 Grimaldi M., 12 Gristina V., 41

Grosso V., 9, 17, 18 Gualandi C., 39 Guariglia R., 21 Guarini A., 22Guarino G., 41 Guerra M., 31 Guglielmi L., 45 , 48 , 49 , 49 Guolo F., 25 Gusolfino D., 7Hambleton S., 32 Heederik D., 18Herbring V., 19Holland S.M., 32 Hsu M., 41 Iacobazzi A., 22 Iacopino P., 22 Iavicoli S., 17Idilli A., 5Imbalzano L., 22 Imbriani M., 12, 39, 435 Indiveri F., 33 Inghirami G., 34 Ingo D., 39 Insalco D., 14 Insidioso M., 38 Intini A., 13 Invernizzi P., 7Irollo E., 44, 46, 49Irrera G., 22Jennrich P., 19 Johnston G., 41 Jönsson BAG., 18 Juris R., 45Kalli F., 7, 33, 34, 37 Klersy C., 29, 33 Kunkl A., 25 Kustrimovic N., 35 La Cava P., 30 La Manna M., 24 La Noce M., 48, 51 La Pietra A., 22 Landi S., 5Lanza F., 7Lari A., 20 Larizza L., 14Laurent S., 35 Lazzari C., 7Legitimo A., 36 Legnaro M., 35 Leonetti C., 46 Leter G., 18, 29 Liga G., 29Lionetti R., 32 Liotta F., 36 Lo Nigro L., 40 Lo Presti E., 24 Locatelli F., 28 Lombardi S., 14 Longo G., 26 Loseto G., 22 Lotti N., 39 Lucarini A., 31 Luchetti F., 8, 31Lucretti S., 5, 9, 17, 18Ludwicki JK., 18Luin S., 5Machiorlatti R., 34 Madonna G., 38 Maggi E., 36

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Maggi Herbring F., 19 Maggi L., 36 Maglia O., 24 Maiello R., 17 Mancardi GL., 37 Mangialetto N., 21 Mannelli F., 20, 26 Mansueto G., 21 Mantelli M., 12Manti A., 9, 16Mantia E., 31 Manzoni M., 41 Marani C., 25 Marcenaro E., 6, 37Marchiani S., 19 Marchini S., 50, Marchionni C., 31 Maresca G., 48, 49 Mariani M., 42 Mariconti M., 33 Marino F., 35 Marone P., 33 Marone U., 38 Marras F., 31, 34 Martelossi S., 37Martinelli G., 8Martini F., 32 Martino M., 22 Martorelli M.C., 21 Masetti R., 28 Materazzi M., 28, 38 Matsuda A., 30 Matturro A., 26 Mazzarese M., 27 , 27 Mazzini G., 6, 11, 12, 13, 39, 41, 42,

43, 46, 47, 50 Mazzoni A., 36 Medico E., 47Menna M., 45 Merchionne F., 22 Merli D., 43 Merlo S., 40, 42, 47Miceli R., 44, 46, 49 Michielotto B., 28 Miglino M., 25 Milanese M., 42 Millo E., 33, 34 Mingari MC., 35 Minoia C., 22 Minzioni P., 12Mirabelli P., 48 Mitra M.E., 24 Monaco A., 39 Montalbano M., 32 Monteleone R., 22 Monti P., 44 Morabito A., 35 Moretta A., 37, 6Moretta L., 31, 37 Mori S., 38 Morra E., 29 Moscato T., 22 Mozzillo N., 38Mulatto P., 33 Muradore I., 45 Muratori M., 16, 19, Mustarelli P., 39, 43 Musto P., 21, 22 Nano R., 46, 50

Pollo B., 47 Pontrelli P., 13 Ponziani V., 26 Porretti L., 42, 51 Porru M., 46 Portararo G., 39 Postuma I., 13Prati D., 51Prato G., 26 Prezzemolo T., 24 Princi D., 22 Profumo A., 43 Proietti M., 34 Prosperi E., 14Protti N., 13 Puliti A., 42 Quaglia S., 37Quaranta V., 14Quartarone E., 39 Queirolo P., 35 Querci V., 36 Quinto I., 51 Ramoino P., 20 Rana A., 22 Rao L., 23 Rasini E., 35 Ratto GM., 5Ravetti J.L., 25 Rebulla P., 42 Rega D., 36 Reguzzoni M., 43 Remuzzi A., 40Ressa G., 22 Ria R., 23 Ricci F., 31, 32 Ricci M., 28, 38 Rigolin G.M., 26 Risi G., 39, 43 Riva F., 43 Rizzati F., 28 Rizzo G., 30 Rocco G., 49Rocco M., 28 Rocco V., 25 Romagnani S., 36 Romano A., 30 Romano M., 50 Rondelli R., 28 Rondelli T., 20 Rosamilio R., 28 Rossetto P., 26 Rossi E., 45 Rossi M., 31 Rossi M.C., 36 Rossi P., 42 Rossini S., 29Rosti V., 50 Rusconi C., 29 Russo A., 38 Sacchi P., 33 Saieva L., 24 Sala S., 24 Sale S., 25 Salis A., 33 Salomone R., 27, 27 Salvati E., 46 Salvi S., 35 Sancilio S., 13 Sanguineti M., 34

Napolitano M., 36 Nardella M., 45, 49, 50 Nardi L., 18 Natali I., 16Nebbioso A., 51 Negrini S., 7, 33Negro A., 5Neri T., 14 Not T., 37Novati S., 33 Novelli G., 45 Nuccorini R., 26 Ogata K., 30 Okere B., 31 Olivieri A., 26 Olivito B., 16, 19, Omes C., 43 Ongari M., 7Orlando V., 24Orofino G., 31 Orsini G., 36 Orzan F., 47 Pacchierotti F., 18, Pace U., 36 Pagliaro P., 31Paino F., 48, 51 Palini A., 23, 44, 45 Palmi C., 29 Palmieri C., 51 Palmieri G., 28, 38 Palumbo G. A., 30 Palumbo G., 21 Pancani F., 26 Pancione Y., 25, Panini N., 47, 50 Paolini A., 46, 50 Papa S., 8, 9,16, 31Papaccio G., 48, 51 Paracchini L., 50 Paredes J.M., 5Parodi A., 7, 31, 33, 34, 37 Parrinello N. L., 30 Pascale S., 26 Pashkoulov D., 18 Pasi F., 46, 50 Pasini A.L., 7Pautasso M., 26 Pedersen HP., 18 Pedrinelli R., 14 Pellegatta S., 47Peruzzi B., 20, 20 Pesce S., 37, 6Pession A., 28 Petecchia L., 43 Petix V., 37Petrini S., 14 Pezzullo L., 28 Piccolini V.M., 12 Picone C., 26, 30, 50 Pietra G., 35 Pietrantuono G., 21 Pinotti P., 7Pirozzi G., 10, 44, 46, 49, 51 Pistillo MP., 35Pittaluga A., 42 Pizzuti M., 26 Poggi A., 35 Pojero F., 27, 27Polla B., 41,

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Santarlasci V., 36 Santin G., 12 Santoni A., 51 Sarno J., 29 Savino A., 29 Savio M., 11Sbrana F., 43 Scaccetti A., 28, 38 Scala D., 36 Scala G., 51Scala S., 36Scalise V., 14 Scampini L., 29 Scappini B., 26 Scarpati B., 29 Schollenberger L., 19 Scovassi A.I., 11, 42, 47 Selleri C., 28 Serio B., 28 Serpero L., 7, 37Serratì S., 22 Sessa G., 44, 46, 49 Silva G., 42, 47 Simeon V., 22 Simeone E., 38 Simeone M., 25 Simonato A., 7Simonazzi G., 31 Siragusa S., 24, 24 Sivori S., 37 Sizzano F., 23, 44, 45 Slatter M., 32 Solimando A., 23 Soverini S., 8 Spagnuolo G., 51 Spanò M., 18 Spedini P., 7Stacchini A., 26 Stasi A., 13 Statuto T., 21, 22 Stigliano A., 45 Stoppacciaro A., 46 Storti AE., 49, 50 Su K., Ko A., 41 Sulis Sato S., 5Surdo S., 42, 47 Tacchetti C., 42 Tagariello G., 23Tagliafierro G., 20 Tajana M., 7Tamburrino L., 19 Tamura H., 30 Tancredi R., 50 Tanzi F., 39 Tardito S., 33, 34 Tarricone M., 25 Tassone P., 17 Tazzari P.L., 31 Te Kronnie G., 29 Tedone E., 34 Tenore A., 26, 30 Terranova V., 36 Tillhon M., 14Tirino V., 48, 49, 51 Tocco MG., 49 Toft G., 18 Tommasini A., 32 Tommasino C., 38 Torchio M., 41, 47

Torre M., 29 Torricelli F., 20Torriglia A., 11Trano A.C., 16Traverso P., 7Triolo A., 30 Trombetta E., 51 Trotta A., 36 Tsukada J., 30 Tuccillo F. M., 51 Ubezio G., 33 Uccelli A., 7, 37 Urbani S., 39 Ursini C.L., 17 Usai C., 20, 42, 43 Vacca A., 23 Vaghi P., 43 Valentini A., 49 Valle A., 44 Vanelli L., 26, 30 Vanzulli A., 29 Vassalli M., 43 Vasta M., 41 Venditti A., 8Veneroni P., 13 Ventura A., 37Vercellino M., 39, 43 Vignoli M., 32 Villa C., 23, 44, 45Villani G., 28 Villani O., 21 Visai L., 12, 39, 43 Visco-Comandini U., 32 Viscoli C., 31, 34 Weldon D., 41 Yamashita T., 30 Yokose N., 30 Zamai L., 8, 31, 34 Zamarchi R., 45 Zangrando A., 28 Zannini C., 31 Zijno A., 18 Zilioli VR., 29 Zizza P., 46 Zonta C., 13Zviezdai V., 18