(ESCCA) and - Wiley Online Library

Cytometry Part B (Clinical Cytometry) 78B:406–459 (2010)

ESCCA and SIC 2010 Abstracts

Abstracts from the 10th Euroconference on Clinical Cell Analysis of the European Society for Clinical Cell Analysis (ESCCA) and the Iberian Society for Cytometry (SIC) PLE-1-01 THE EU-FUNDED EUROFLOW PROJECT (LSHB-CT-2006-018708): BACKGROUND, CURRENT ACHIEVEMENTS AND FUTURE PERSPECTIVES Jacques J.M. van Dongen,1 Alberto Orfao2 1

Erasmus MC, ROTTERDAM, The Netherlands 2 Cytometry Service, Dept of Medicine & Cancer Research Centre, University of Sala, SALAMANCA, Spain Flow cytometric immunophenotyping is widely used for the diagnosis, classification, and monitoring of hematological malignancies. However, molecular techniques have a strong position for classification and monitoring of residual disease, despite several major disadvantages, such as their labor-intensive and time consuming character, limited applicability and no focus on cell populations within a sample, unless preceding purification steps are performed. These limitations can be overcome by innovations in flow cytometry immunophenotyping because this technology fulfills the requirements of high speed, accurate focusing on malignant cell population as well as broad applicability for the diagnosis and follow-up of virtually all hematological malignancies. Consequently, the EuroFlow Consortium worked for 4 years on several Work Packages with special focus on: 1) the development of new antibodies for the detection of intracellular (onco)proteins, 2) application of a novel immunobead technology for fast and easy detection of fusion proteins for classification of acute leukemias, 3) the development of new software tools for easy and reproducible data analysis, 4) the design and evaluation of 8-color antibody panels and instrument set-up procedures for the diagnosis, classification and monitoring of patients with different types of hematological diseases. The combination of panels and software tools allows easy evaluation of the immunophenotypic profile of a given cell population against reference data of multiple normal and pathological samples stained with the same antibody panels. This work was performed by 14 academic diagnostic teams and 2 small-sized biotech companies.

PLE-1-02 EUROFLOW DESIGN OF SOFTWARE TOOLS TO BUILD MULTICOLOR ANTIBODY PANELS FOR LEUKEMIA/LYMPHOMA PHENOTYPING Alberto Orfao,1 Quentin Lecrevisse,1 Elaine S. Costa,2 Carlos E. Pedreira,3 Marta Martin,4 Juan Hernandez,4 Miguel Munõz,4

q 2010 International Clinical Cytometry Society

Juan Flores-Montero,1 Julia Almeida,1 Sebastian Bottcher,5 Ludovic Lhermite,6 Tomas Kalina,7 Lukas Sedek,8 Vincent H.J. van der Velden,9 Matthew Cullen,10 Paulo Lucio,11 Jacques J.M. van Dongen,9 EuroFlow Consortium12 1

University of Salamanca, SALAMANCA, Spain Instituto de Pediatria e Puericultura Martagaõ Gesteira and Departamento de Clıń, RIO DE JANEIRO, Brazil 3 Faculty of Medicine and COPPE - Engineering Graduate Program, UFRJ/Federal Univ, RIO DE JANEIRO, Brazil 4 Cytognos SL, SALAMANCA, Spain 5 University Klinik Scheswig-Holstein, KIEL, Germany 6 Hopital Necker, PARIS, France 7 Charles University, PRAGUE, Czech Republic 8 Medical University of Silesia, ZABRZE, Poland 9 University Medical Center Rotterdam, ROTTERDAM, The Netherlands 10 St James University Hospital, LEEDS, United Kingdom 11 Instituto Portugues de Oncologia, LISBON, Portugal 12 Spain In recent years, the EuroFlow Consortium has developed new and unique tools which can be applied to a more comprehensive and objective evaluation of the performance of multicolor antibody panels, for example, leukemia/lymphoma phenotyping. The new strategy is based on the transformation of a set of 2 data files containing information about the same sample into a single data file. If the merged data files correspond to different stainings of the same sample containing common backbone parameters that allow unequivocal identification of the cell populations of interest in that sample, merging of the data files can be followed by an accurate calculation of the ‘‘missing values’’. As a result, all individual events from each of the original data files will contain information about each reagent in the whole panel evaluated in all aliquots stained for the same sample. Based on this strategy, one or more cell populations from one or multiple samples can now be compared with other (e.g. reference) cell populations from one, or a pool of 2 different samples. To a certain extent, this allows objective comparison of the immunophenotypic profiles 2

Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.b.20563

ESCCA AND SIC 2010 ABSTRACTS

obtained with a given panel of reagents in a sample with the profiles obtained for the same combinations of reagents in another sample (or groups of samples) composed of normal, reactive, activated or malignant cells. For this type of comparison multivariate statistical analyses as those provided by Principal Component Analysis. Through such comparisons, one can easily evaluate the performance of antibody panels to discriminate among two or more groups of diseases or between normal/reactive vs neoplastic conditions.

PLE-2-01 MATHEMATICAL MODELING OF IMMUNOFLUORESCENCE DISTRIBUTIONS Francesco Lampariello

IASI-CNR, ROME, Italy The definition of a positively or negatively stained cell population by flow cytometric techniques is discussed. The attention is focused on the analysis and interpretation of histograms with weak marker expression appearing in the majority of hematological malignancies. The simple separation of positive from negative cells with a cut-off marker leads to subjective and controversial results, but is reported just as ‘customary’ in literature. The Kolmogorov-Smirnov (KS) statistical test can be used for the quantitative histogram comparison of a cell sample and the control. From the application of the KS test to a set of replicate controls, it has been shown that even differences due to the sample preparation and/or to instrumental variation may be considered significant. This outcome indicates the need for determining an appropriate estimation of the overall variability in the control data, thus allowing to distinguish between statistical and biological significance of the differences.Therefore the KS statistics can be used as a reference for determining this limit. Finally, for the analysis of distributions where the positive percentage is of interest, the main features of the two different methods are briefly described. The first one is based on the use of a suitable statistical model for mathematically representing both the homogeneous (positive and negative) cell populations and hence the whole test histogram. The second one, easier to implement, relies on the analysis of the ratio of the cumulative distributions associated with the test and control histograms. By analyzing a set of experimental data, both methods appear to be effective in the entire range of the percentage values.

PLE-2-02 A FUNCTIONAL FRAMEWORK FOR TOTAL MDS (DIGITAL) MANAGEMENT John Drakos,1 Katerina Psarra,2 Marina Karakantza1 1

University of Patras, PATRAS, Greece Evangelismos Hospital, ATHENS, Greece Myelodysplastic Syndromes (MDS) represent a group of clonal stem cell disorders with significant heterogeneity regarding clinical presentation, progression and response to treatment. The heterogeneity of MDS reflects the complexity of the underlying pathogenetic mechanisms, as well as the limitations of the available diagnostic methods.

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The WHO classification and the international prognostic systems have not incorporated immunophenotyping in the diagnostic and prognostic criteria for MDS. However, the published experience indicates that FC results significantly improve the diagnostic and prognostic efficiency. The main purpose of our work is to introduce a new approach to the storage, management and interpretation of MDS data in order to upgrade the diagnostic and prognostic significance of available laboratory and clinical information and to improve the correlation of experimental results to the clinical and laboratory characteristics of the MDS patients. To achieve our research goals we focused on: 1) an expertly combined analysis of the particular characteristics of FC, cytogenetic, molecular, morphological and clinical data, 2) the development of optimal solutions for storage, management and integrated data analysis. The result of our work was a functional framework for the total digital management of the disease that enables: – evaluation of the known diagnostic and prognostic parameters, – identification of new diagnostic and prognostic parameters, – identification of new pathogenetic mechanisms, – identification of subgroups of patients with common clinical characteristics and pathogenetic mechanisms, – identification of subgroups of patients responding to therapeutic agents.

PLE-2-03 A PROBABILISTIC APPROACH TO ANALYSE MINIMAL RESIDUAL DISEASE IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA Alberto Orfao,1 Elaine S. Costa,2 Carlos E. Pedreira,3 Quentin Lecrevisse,1 Susana Barrena,1 Antonio Lopez,1 Sandra Quijano,1 Marta Martin,4 Carlos Fernandez,1 Juan Hernandez,4 Miguel Munõz,4 Juan Flores-Montero,1 Sebastian Bottcher,5 Jacques J.M. Van Dongen,6 EuroFlow Consortium7 1

University of Salamanca, SALAMANCA, Spain Instituto de Pediatria e Puericultura Martagaõ Gesteira and Departamento de Clıń, RIO DE JANEIRO, Brazil 3 Faculty of Medicine and COPPE - Engineering Graduate Program, UFRJ/Federal Univ, RIO DE JANEIRO, Brazil 4 Cytognos SL, SALAMANCA, Spain 5 University Klinik Scheswig-Holstein, KIEL, Germany 6 University Medical Center Rotterdam, ROTTERDAM, The Netherlands 7 Spain Over the last decades, important advances have been achieved in the treatment of B-cell chronic lymphocytic leukemia (CLL) with increasingly high complete remission rates. Thus, a demand for more sensitive techniques to detect low numbers of residual CLL cells after therapy has become a challenge. Among other approaches, multiparameter flow cytometry immunophenotyping (MFCI) has become the preferred method for minimal residual disease 2

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(MRD) monitoring in CLL after therapy. This is mainly due to the many advantages of MFCI and the unique aberrant features of CLL cells. The utility of MFCI-based MRD monitoring of CLL is further supported by the demonstration of its sensitivity and clinical impact in an increasingly high number of single-center studies reported in the literature. Recently, we have proposed and evaluated a probabilistic approach based on pattern classification tools and the Bayes theorem, for automated analysis of MFCI data from a relatively large group of CLL versus normal peripheral blood B-cells under MRD conditions, to reduce operator-associated subjectivity. The proposed approach provided a tool for MRD detection by MFCI in CLL. The sensitivity was systematically < 1x10-4 (median of < 10-7). Thus this approach, based on the search for minimal numbers of neoplastic B-cells similar to those detected at diagnosis, could potentially be applied with both a high sensitivity and specificity to investigate MRD in virtually all CLL patients, using a common consensus single, e.g. 8-color staining.

PLE-3-01 BASOPHIL ACTIVATION TEST BY FLOW CYTOMETRY: PRESENT AND FUTURE APPLICATIONS IN ALLERGOLOGY Didier Ebo

University of Antwerp, ANTWERP, Belgium The diagnosis of allergic reactions in clinical practice rests upon both the clinical history and the demonstration of specific immunoglobulin E (sIgE), either in serum or via skin tests. However, for various reasons, correct identification of the offending allergen(s) is not always straightforward. In an attempt to find reliable methods to investigate hypersensitivity reactions, histamine and sulfidoleukotriene release tests have long been introduced. However, relatively few comprehensive quality reports have been published so far. Upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies in the basophil activation test (BAT). Today, the BAT has been adopted in the diagnosis of IgEmediated allergy to inhalant allergens, drugs, food, natural rubber latex and hymenoptera venom. This review focuses on some of the major clinical applications of flow-assisted analysis of in vitro activated basophils. For further reading: Cytometry B Clin Cytom. 2008 Jul;74(4):201-10.

PLE-3-02 THE BASOPHIL ACTIVATION TEST IN THE DIAGNOSIS OF ALLERGY: TECHNICAL ISSUES AND CRITICAL FACTORS Gunter Sturm

Department of Dermatology, Medical University of Graz, GRAZ, Austria The first protocols for the CD63 based basophil activation test (BAT) were developed in the mid-1990s. Since then, the BAT has been evaluated in numerous studies for

a variety of allergens. Usually, the BAT works well with all kinds of protein allergens, but it is still not optimized for haptens, such as in drug hypersensitivity. The CD63 based BAT is now generally accepted as an additional and reliable diagnostic tool in the diagnosis of immediate allergy. The BAT has been shown as a useful test in difficult cases, especially in hymenoptera venom allergy,. Another common activation marker with different time kinetics, but with similar performance is CD203c, where the first CD203c-based protocol was published in 2001. BAT protocols are generally not standardized; diverse protocols are prone to yield different results. Flow cytometric quantification of activated basophils can be used either on whole blood or on basophils separated by buffy coat centrifugation or sedimentation over dextran. Nevertheless, there is a clear preference for whole-blood assays, which preserve basophils to a greater extent and can be performed more efficiently. Moreover, a large number of gating strategies to characterize basophils are currently in use (e.g. IgEpos, CD123pos/HLA-DRneg, CD3neg/CRTH2pos, CCR3pos). Furthermore, the accuracy of results when BAT is performed with samples stored for several hours or even overnight has been controversially discussed. Another important question is whether or not the shipment of blood samples over several hours to specialized laboratories leads to a loss of basophil reactivity. To solve these issues, the European consortium EuroBAT was founded with the aim of setting shared standards.

PLE-3-03 MECHANISM OF DRUG INDUCED ALLERGY Werner J. Pichler, MD,

University of Bern, BERN, Switzerland Drug allergy is a common clinical problem, which remains elusive for diagnostic tests. To understand drug allergy and to improve the urgently needed diagnostic capabilities, one has to address two main questions: 1) how does a drug become stimulatory for the immune system? and 2) how may one explain the great heterogeneity of drug-induced allergic diseases? – Drugs are small molecules and are not per se immunogenic. Nevertheless, many drugs can induce allergic reactions, sometimes mediated by drug specific IgE, but more often by drug specific T-cells. To gain immunogenicity, drugs need to bind covalently to larger proteins, which are thereby modified and gain antigenicity. Many drugs are unable to bind covalently to a protein, but may gain this ability by drug metabolism. Then the metabolite is immunogenic. – The ensuing drug specific immune response is dependent on the type of molecule modified by the drug, meaning that a different immune mechanism evolves, if a soluble or cell bound molecule is modified. Modification of cellular molecules may also induce a cytotoxic immune response, which is very common in drug hypersensitivity.



– An alternative possibility for drugs to stimulate the immune system is because of their pharmacological activity: drugs are designed to bind to some enzyme pockets, and may thus also bind to some of the highly polymorphic immune receptors like T-cell receptors. In certain circumstances, a rather restricted immune response may arise, e.g. an exclusive T cell response, but only if some T cell receptors/T cells interact with the drug. This unusual stimulation of immune cells is called pharmacological interaction with immune receptors (p-i concept). It is actually the explanation that drugs can stimulate immune cells in vitro, even in the absence of drug metabolism. These features now allow us to use various in vitro assays to detect drug reactive immune response: Different applications of cytometry seem to be suitable: widely used is the basophil activation test (BAT). Stimulation is assumed to occur via crosslinking drug specific IgE on Fc-IgE RI. This can be measured by upregulation of CD63 or other cell surface molecules by flow cytometry. However, how the drug binds to larger molecules able to crosslink Fc-IgERI has not yet been clarified. For the frequent T cell response in drug hypersensitivity, drug specific activation can also be measured by simple flow cytometry (e.g. CD69 upregulation on drug specific cells after 24-72hrs co-incubation with the drug). Since the activation of the few drug reactive T cells induced a substantial bystander activation of other T cells, this assay allows reliable detection of reactive T cells, in spite of limited precursor cell frequency. Alternatively, cytokine production can be measured intracellularly after permeabilizing the T cells or after secretion, e.g. by a beads assay. All these assays are promising, but still need improvement regarding sensitivity. A crucial aspect to enhance the limited sensitivity seems to be an optimal cell culture condition and suited drug preparations.

PLE-5-01 MULTI-COLOR IMAGING FLOW CYTOMETRY FOR CHARACTERIZATION OF CIRCULATING ENDOTHELIAL CELLS J Philip McCoy, Leigh Samsel, Kimberly Woodhouse, Pradeep K Dagur, Nalini Raghavachari

National institutes of Health, BETHESDA, MD, United States of America Circulating Endothelial Cells (CECs) are angiogenic cells that are increased in the peripheral circulation as a result of vascular injury or in response to angiogenic stimuli. CECs have been detected by immunomagnetic bead selection, microscopy or flow cytometry. Flow cytometry provides quantitative information regarding the number of CECs, but offers no morphology. Microscopic studies of CEC morphology are few and the results varied. Few studies have validated the immunophenotypic identification of CECs with molecular techniques. Multi-color imaging flow cytometry (MIFC) combines traditional flow cytometry and microscopy, and is suitable for rare event analysis such as CECs. In the current study, CECs from patients with sickle cell disease (SCD) are identified by MIFC and the identity of


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these cells has been confirmed by Q-PCR using parallel sorting of the same sample. PBMCs from patients with SCD were stained with CD146, CD3, and CD45. Hoechst and 7AAD were used to identify cell nuclei and dead cells, respectively. One aliquot was analyzed on the Amnis ImageStreamX imaging cytometer and a second aliquot was sorted on a MoFlo cytometer. Single, live, nucleated CD146þ, CD3-, CD45- cells with appropriate light scatter were identified, and the morphology and patterns of marker staining examined. On sorted CECs, Q-RTPCR identified mRNA encoding CD146, caveolin, eNOS, and heme oxygenase-1, all known to be expressed on endothelial cells. mRNA for CD3 or CD19 was not detected. This technology should permit further studies on CEC characteristics and function that are not possible using other techniques, and may also provide further insight into the use of CECs as biomarkers.

PLE-5-02 SIGNATURE PROFILES OF CMV-SPECIFIC T-CELLS IN PATIENTS WITH CMV REACTIVATION AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION Tomas Kalina,1 Ladislav Kro´l,1 Jan Stuchly´,1 Petra Keslova´,2 Petr Hubaćek,2 Petr Sedlaćek,2 Jan Stary´,2 Hrusˇa´k Ondrej1 1

CLIP-Cytometry, Dpt of Pediatr. Hematol. / Oncology, Charles University, PRAGUE, Czech Republic 2 Dept of Pediatr. Hematol. / Oncology, Charles University, PRAGUE, Czech Republic Depletion of cellular immunity as a consequence of conditioning before allogeneic hematopoietic stem cell transplantation frequently results in CMV reactivation which may, in turn, lead to life-threatening infections and require timely antiviral treatment. We have investigated the functional signatures of CMV-specific CD4þ and CD8þ T-cells in 191 samples from 118 individuals. We included patients with either high or undetectable viral loads and those who controlled or did not control their CMV reactivations. All patient subsets were compared to healthy donors. Polychromatic flow cytometric measurements of CD154 (CD40L), intracellular cytokines (IFN, IL2), and a degranulation marker (CD107a) revealed the functional status of various T-cells simultaneously. We found that dual IFN/IL2 producing CD8þ T-cells were significantly decreased in patients who did not control their CMV reactivations compared to those who did. In contrast, CD8þ T-cells that produced IFN only were the most abundant subtype but they were present in a substantial number of non-controllers. Hierarchical clustering of distinct functional signatures revealed that polyfunctional CD8þ T-cells were acting in concert with other subsets, whereas the isolated production of IFN by CD8þ T cells showed insufficient collaboration with others. In conclusion, our study revealed functional signs that may be useful for immune monitoring, which may change the interpretation of previous studies that assessed only IFN. The project was supported by Ministry of Health of Czech Republic grant No. NS /9996, MSMT21620813 and MZO FNM2005

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PLE-5-03 NFKB TRANSLOCATION AS A QUANTITATIVE PARAMETER FOR CLINICAL IMMUNOSUPPRESSION Hans Minderman,1 Oleh Pankewycz,2 Kieran O’Loughlin,1 Lin Feng,2 Mark Laftavi,2 Paul Wallace1 1

Roswell Park Cancer Institute, BUFFALO, NY, United States of America 2 Buffalo General Hospital, BUFFALO, NY, United States of America Individualized optimal dosing of immunosuppressive therapy requires an assay that, using quantifiable markers of diagnostic and therapeutic responses, monitors immune function.. Cell-mediated immunity is regulated by key signaling pathways of which the intracellular distribution of specific intermediaries correlates with signaling activity and, therefore, cellular function. This is quantifiable in immunophenotypically-defined cells with the ImageStream platform. Peripheral blood from healthy volunteers and transplant recipients undergoing treatment with tacrolimus, mycophenolic acid and prednisone were tested for the ability of stimulants (TNF and PMA/ionomycin) to induce translocation of NFkB in lymphocyte subsets (CD3, CD4, CD8). A socalled similarity score algorithm was used as a continuous variable quantifying the nuclear translocation of NFkB (p65) on an individual cell basis on at least 5,000 cells/sample. Using this parameter, a clear quantitative difference was observed between the ability of the stimulants to induce NFkB nuclear translocation ex-vivo in individuals undergoing immunosuppressive treatment versus healthy donors. Furthermore, preliminary data indicate that this parameter correlates with the in vivo immune response with respect to clinical presentation (stable presentation, rejection or infection) and has an improved concordance with clinical presentation compared to the Immuknow assay which uses the rise in cellular ATP levels in CD4 cells following ex-vivo stimulation as a parameter for immune response. Supported in part by NIH 1R21CA126667, 1S10RR022335-01, and Cancer Center Support Grant CA016056.

PLE-5-04 T CELL EPITOPE DISCOVERY THROUGH HLA CLASS I PEPTIDE LIGAND EXCHANGE AND COMBINATORIAL CODING Wim van Esch,1 Juk Yee Mok,1 Annemieke Molenaar,1 Arnold Bakker,2 Mireille Toebes,2 Sine Reker Hadrup,2 Chengyi Shu,2 Ernst Soethout,3 Josine van Beek,3 Huib Ovaa,4 Ton Schumacher2 1

Sanquin Reagents, AMSTERDAM, The Netherlands Div. Immunology, The Netherlands Cancer Institute, AMSTERDAM, The Netherlands 3 Netherlands Vaccine Institute, BILTHOVEN, The Netherlands 4 Div. Cellular Biochemistry, The Netherlands Cancer Institute, AMSTERDAM, The Netherlands Human major histocompatibility complex class I (HLAI) consists of beta2-microglobulin, a heavy chain and a pep2

tide ligand. Peptides in context with HLA-I on the cell surface are recognized by appropriate cytotoxic T cells. Our aim is to develop a platform for high-throughput identification of HLA-I ligands and T cell detection using flow cytometry in order to detect new disease-specific epitopes/T cells. To this end, conditional ligands that can be cleaved in the HLA-bound state upon UV irradiation were designed for different HLA alleles. Cleavage of HLA-I-bound conditional ligand in the presence of another experimental ligand results in peptide exchange. Only those peptides that fit in the peptide binding groove will be able to maintain the integrity of the empty HLA complex. Efficiency of HLA-I stabilization by ligands can be determined by ELISA. Subsequently, HLA-I-binding peptides identified in this manner are tested for their immunological relevance. For this purpose, combinatorial coding technology has been developed that allows the parallel detection of a multitude of different T cell populations in a single sample. In this way, comprehensive screens can be performed. Using these technologies, several screens for HLA ligands have been performed, either manually or using robotics, using peptides potentially associated with (non-)infectious diseases. Large-scale screenings of their immunological relevance by analysis of T cell responses using combinatorial coding are ongoing. Together these techniques form a highly efficient platform for T cell epitope discovery in a wide range of human diseases and for the monitoring of disease- and therapy-induced T cell immunity.

PLE-5-05 FACS-SORTED B-CELL SUBPOPULATIONS FROM NORMAL HUMAN SECONDARY LYMPHOID TISSUE; FIVE SUBSETS WITH CONVERGENT GENE EXPRESSION PROFILES AND PHENOTYPE Alexander Schmitz,1 Malene Kragh Kjeldsen,2 Martin Perez-Andres,3 Preben Johansen,4 Kirsten Fogd,2 Martin Boegsted,2 Mette Nyegaard,2 Alberto Orfao,3 Hans E. Johnsen,2 Karen Dybkaer2 1

Department of Haematology, Medical Center & AHSIC, Aarhus University Hospital, AALBORG, Denmark 2 Department of Haematology, Medical Center and Aalborg Hospital Science and Innov, AALBORG, Denmark 3 Service of Cytometry & Department of Medicine, CICancer-University of Salamanca, SALAMANCA, Spain 4 Department of Pathology, Aalborg Hospital, AALBORG, Denmark Background: At present, B cell differentiation and development are recognized as being very complex. High speed multiparameter flow cytometry (MFC) and cell sorting (FACS) enable fast and sensitive identification and characterization of normal and malignant B-cell hierarchy. Hypothesis and Aims: We hypothesize that B-cell subpopulations identified by MFC have distinct gene expression profiles reflecting their global functions. The aim is to use MFC, sort identified B-cell subpopulations for gene expression profiling (GEP) to gain a better understanding of normal function and differentiation.



Methods: From homogenized human tonsil tissue, isolated mononuclear cells are subjected to MFC and FACS-sorted using a multicolor fluorescence single tube (CD3/CD10/ CD20/CD27/CD38/CD44/CD45/CXCR4), to identify and isolate distinct B-cell subpopulations for morphological inspection, and both global and single gene expression profiling. Results: Five B-cell subsets from human tonsil, namely naı¨ve, centroblast, centrocyte, memory and plasmablast have been identified, as well as FACS-sorted based on their distinct immunophenotypic features. The cellular identity of the subpopulations was verified at the gene expression level using microarray and qRT-PCR gene expression profiling based on the used discriminative phenotypic markers as well as transcriptions factors like KI-67 and multiple B-cell differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, X BP1). Conclusion: Using a combination of surface markers expressed antigens and gene expression analysis of B cell subsets, a strong methodology is provided to generate improved insights into the B-cell biology and thereby also the development of B-cell malignancies.

PLE-5-06 MENINGIOMAS WITH DIFFERENT CYTOGENETIC PROFILES DISPLAY DISTINCT IMMUNOPHENOTYPIC FEATURES AND VARIABLE PERCENTAGES OF INFILTRATING NON-TUMORAL ANTIGEN-PRESENTING CELLS Patrıćia Domingues,1 Cristina Teodo´sio,1 Marıá Dolores Tabernero,2 Paloma Ba´rcena,1 Maria Carmen Garcıá-Macias,3 Javier Ortiz,3 Frans Bom,1 Julia Almeida,1 Alberto Orfao1 1

Centro de Investigacion del Cancer, SALAMANCA, Spain IECSCYL - Hospital Universita´rio de Salamanca, Spain 3 Servicio de Anatomia Patolo´gica, Hospital Universita´rio de Salamanca, Spain Meningiomas are genetically heterogeneous primary tumors of the central nervous system. Few studies have been reported in which their immunophenotype have been characterized in detail. Therefore, we analyzed the immunophenotypic features of meningiomas and it relationship with tumor cytogenetics. Meningiomas (n¼42) were studied in a broad spectrum of flow cytometry (MFC) markers, after staining with DRAQ5, and fluorescence in situ hybridization (FISH). Phagocytosis/endocytosis studies were performed on 9 tumors, also FACS-sorted for Papanicolau staining and FISH. MFC revealed coexistence of tumor cells (TC) and nonTC populations in all meningiomas. TC displayed significantly higher expression of CD13, CD58 and HLA-I among diploid cases; in turn, higher levels of bcl2 were detected in cases with isolated monosomy 22/del(22q) (-22), whereas cases with complex karyotype had decreased expression of HER2-neu and CD55 together with higher proliferation rate. Non-TC populations included lymphocytes and antigen presenting cells (APC), with HLA-DRþ/CD14þ/CD16-/þ/ CD45þ/CD68þ phenotype and phagocytic/endocytic ability. APC were significantly increased among -22 meningiomas. Our results show that meningiomas with distinct cytogenetic profiles display different immunophenotypic features along with higher proliferation index in complex 2


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karyotype cases and greater infiltration by APC among -22 meningiomas, indicating the existence of different interactions between TC and the immunological microenvironment in meningiomas with distinct cytogenetic profiles. Grants: FCT-Portugal (PIC/IC/83108/2007; SFRH/BD/ 64799/2009); ISCIII-Spain (FIS/FEDER06/0312; RTICC RD06/0020/0035); Caja Burgos.

PLE-6-01 GLOBAL CYTOMETRY NETWORKING: THE DREAM BECOMES REALITY Maria Arroz

CHLO, Hospital S. Francisco Xavier, LISBON, Portugal This lecture will cover 2 main topics: the European Clinical Cytometry Courses and immune activation in HIV disease. The evolution of the 5 Clinical Cytometry Courses (ECC) will be presented, with relevance to dates, places, number of participants and countries of origin, and evaluation of the Faculty. A major feature of the ECC has been the concept of continuous education, offering at least one different option each year. This has proven to be successful in the sense that some participants have attended several courses from the first year onwards. It has also been our policy that the Faculty includes both European and USA experts. A cohort of 58 HIV infected patients without antiretroviral therapy (ART) was followed for eight years. During that period of time 30 patients began ART. The aim of this study was to correlate the expression of some molecules on the membrane of CD4þ and CD8 þ T cells, namely the activation marker CD38, CD11a and CD45 RO to define naı¨ve and memory T cell subsets and CD57 as an effector antigen on cytotoxic T cells. The data demonstrates that replication of the virus is related to the activation of the immune system as evidenced by the expression of CD38 on the CD4þ, CD8þ T cells and monocytes and that this activation occurs in parallel in the different types of cells. The naı¨ve and memory subsets also tend to go together in CD4þ and CD8þ T cells throughout disease progression, following some sort of homeostatic mechanism. Finally, CD8þ T cell subsets seem to be more involved in the immediate immune reconstitution following ART, the activation of the CD4þ T cells taking longer to attain the levels observed in the controls.

PLE-7-01 IMPACT OF CMV CARRIER STATUS ON CIRCULATING T CELLS Paul Moss

University of Birmingham, BIRMINGHAM, United Kingdom Cytomegalovirus (CMV) is one of the human herpes viruses and infects most of the population. It is an important pathogen in immune suppressed patients and there is

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now considerable interest in its potential role in other clinical situations. Perhaps the most remarkable feature of CMV is its level of extreme immunodominance. Primary infection stimulates the development of a very large CMV-specific CD4þ and CD8þ T cell immune response and, as the virus is never cleared, this memory response must be maintained throughout life. Indeed, evidence is now emerging that this cellular response can increase in magnitude during ageing and may contribute to the development of immune senescence. The phenotype and function of CMV-specific T cells is quite unique. Both the CD8þ and CD4þ compartments have a cytotoxic function and are highly differentiated with frequent loss of CD27 and CD28. They also often show ‘reversion’ to expression of CD45RA and begin to express molecules that are associated with NK cells, such as CD57 and KIR. The magnitude of the CMV-specific CD8þ T cell response is startling and may comprise 10-40% of the total CD8þ compartment. CMV-specific CD4þ T cells might represent 3-10% of the CD4þ pool. These changes are so marked that the global T cell repertoire is permanently changed following CMV infection. Indeed, it may be argued that it is impossible to interpret the immune system in disease states without knowledge of the CMV status of the patient. The presentation will discuss the generation, function and phenotype of the CMV-specific immune response and indicate how flow cytometry has played an important role in exploring this unique host-virus relationship.

have different functions and their repertoires are shaped by different selection pressures.

PLE-7-03 CAN AGEING BE DELAYED? Calogero Caruso, Giuseppina Candore, Giuseppina Colonna-Romano

University of Palermo, PALERMO, Italy The data on the determinants of human longevity indicate that centenarians are a good choice for the study of longevity, because they represent an extreme phenotype, i.e., the survival tail of the population who escaped neonatal mortality, pre-antibiotic era illnesses and fatal outcomes of age-related complex diseases. Centenarians are quite capable of mounting effective inflammatory responses; however, inflammatory status is compensated by the concomitant development of strong and effective anti-inflammatory responses. Hence, the study of centenarians allows us to correlate longevity and some of the major age-related pathologies, known to be inflammtion-related. Centenarians are remarkably enriched in ‘‘good’’ genotypes and show opposite frequencies of ‘‘bad’’ genotypes in comparison with patients affected by major age-related pathologies, such as atherosclerosis, Alzheimer’s disease and cancer. Therefore, they can be considered as ‘‘super-controls’’, allowing a reliable and accurate identification of the variants related to the major killer and as further proof that longevity is, at least in part, the positive counterpart of unsuccessful ageing. However, data on centenarians might contribute to the determination of a risk profile which may allow both the early identification of individuals susceptible to disease and the possible discovery of potential targets for drugs.

PLE-7-02 B CELLS AND AGEING Deborah Dunn-Walters

PLE-8-01

King’s College London, LONDON, United Kingdom Age-related changes in the structure and function of the immune system, collectively termed immunosenescence, result in poor responses to infections, increased susceptibility to cancers and increased incidence of autoimmune diseases. The humoral immune response, maintained by the B cell compartment, plays a key role in an effective immune system. However, there is an age-related decline in the effectiveness of antibodies produced in response to infection or vaccination. A diverse B cell repertoire is deemed to be essential for a healthy immune system, not only to provide a variety of good quality antibodies to recognise the multiplicity of likely pathogen challenge, but also because B cells are important regulators of the immune response. In addition to their excellent capabilities as antigen presenting and activating cells, recent work shows that some sub-populations of B cells can have suppressive functions. We have shown that the diversity of the B cell population as a whole decreases with age, and is associated with ill health. Whether decreased diversity is a feature of all B cells or a reflection of altered sub-populations is not clear, since we have also shown that different subsets of B cells

THE BIOLOGY OF MBL VS CLL: DIFFERENCES AND SIMILARITIES Paolo Ghia, Claudia Fazi, Antonis Dagklis, Lydia Scarfo`, Cristina Scielzo

Universita` Vita-Salute San Raffaele and Istituto Scientifico San Raffaele, MILANO, Italy Monoclonal B lymphocytes can be found in the peripheral blood (PB)of healthy individuals, at a concentration 50% in people older than 90 years.



Monoclonal B cells can be present at strikingly different cell concentration ranging from 1-50cells/L to 1.54.9x106/L). The B cell concentration in the PB has clinical bearing as the cases with higher concentration may more frequently evolve into a CLL, though still at a low frequency (1-2%/year). Though the latter cases may be considered a pre-leukemic condition, most MBL, especially those with low cell concentrations, may simply reflect the progressive senescence of the immune system as suggested by the higher prevalence of MBL when compared to CLL (at least 100 times less frequent) and the increasing frequency with age. This is also suggested by the molecular features displayed by ‘‘low-count’’ MBL that may alsobe polyclonal/oligoclonal, perhaps following continuous exposure to persistent autoor foreign antigens.

PLE-8-02 CELL COUNTING, HISTORY OF MBL AND THE CHANGING DIAGNOSIS OF CLL Gerald E. Marti

FDA, Silver Spring, MARYLAND, USA Based on the work of Galton and Hanson, the Rai clinical staging system relied on an ALC of 15 109/L for the diagnosis of CLL. In 1988 and 1996, the NCI-WG required 5 109/L ALC with flow cytometric detection of clonal B cells via light chain restriction. MBL was defined as a persistent monoclonal lymphocytosis with an absolute B cell count of < 5 109/L. In 2008, the iwCLL made the diagnosis of CLL based on an ALC of 5,000 B cells. The history of MBL can be divided into three areas: population studies, clinical lymphocytosis, familial and sporadic CLL. Shim (SupraFund toxic waste sites environmental exposure population studies); Rachel (MBL in the adult blood donor population); Ghia (geographically isolated population northern Italy); and Orfao (healthy volunteers in a region surrounding Salamanca, Spain). Three observations have emerged: (1) prevalence is dependent upon the method; (2) MBL increases with age and (3) the prevalence of MBL is greater than the prevalence of CLL. Rawstron has described the pattern of MBL with lymphocytosis, its absolute B cell count (BALC) and progression to CLL in clinic outpatients. These studies serve to differentiate the low count MBL where the BALC is normal and clinical subjects with a B cell lymphocytosis. The percentage of B cell clone MBL in each of these settings can vary from less than 5% to greater than 95%. In a retrospective study of cyropreserved whole blood samples, Landgren has shown that all patients who developed CLL have preclinical MBL associated with an imbalance of serum free light chains. The finding of MBL in unaffected first-degree relatives in kindred with familial CLL is perhaps the most interesting, from a genetics point-of-view, but it is complicated. Initially, these individuals were thought to have the higher prevalence MBL (approx 15%). In retrospect, this group of individuals also provide an opportunity to consider HLA-matched related donor transfer of MBL.


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PLE-8-03 NATURAL HISTORY OF CLINICALLY RECOGNIZED MONOCLONAL B-CELL LYMPHOCYTOSIS (MBL) VERSES RAI STAGE 0 CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) Tait D. Shanafelt

Mayo Clinic, ROCHESTER, MN, USA In 2008, the diagnostic criteria for chronic lymphocytic leukemia (CLL) were changed from being based on the absolute lymphocyte count (5.0 109/L), to the absolute B-cell count (5.0 109/L). Although these criteria expanded the number of patients being classified as having monoclonal B-cell lymphocytosis (MBL), there was limited information on the clinical implications of this distinction. In 2009, we reported a longer treatment-free survival (TFS) of individuals with clinically recognized MBL (n¼302) as compared to patients with Rai stage 0 CLL (n¼329) [JCO 27:3959]. Nonetheless, in a separate analysis of 459 patients with a clonal population of CLL phenotype, we found that a B-cell threshold of 11x109/L was a better predictor survival than the currently used threshold used to distinguish between MBL and CLL (5.0 109/L) [Blood 113:113:4188]. Despite the importance of these findings, the follow-up time for the patients in these series were relatively short (median 34 months and 18 months, respectively). We have now updated the follow-up of the patients in both series to provide further insight into differences in the natural history of patients with clinically recognized MBL when compared to Rai stage 0 CLL. The results of the updated analysis will be presented and discussed.

PAR-1E-01 RELATIONSHIP BETWEEN CD34 NEGATIVE VERSUS CD34 POSITIVE HUMAN HEMATOPOIETIC STEM CELLS. Dominique Bonnet

Cancer Research UK, LONDON, United Kingdom A rare CD34neg cells with SCID-Repopulating (SRC) activity exists (Lin-CD34-CD38-; -/-) and, using in vivo serial transplantation assays, we can demonstrate that CB -/- cells are more primitive, as they not only generate þ/- HSCs in vivo and, in the long-run, are more competent than the conventional CB þ/- cells. Detailed characterization of these two populations showed all CB -/- cells are in quiescent stage. Multiple mRNA and protein expression studies confirm this. Moreover, the lower expression of Lmo2, Runx1A and Scl/Tal1 in -/- cells compared to þ/- cells suggests that they are immature cells. We uncovered a uniquely high expression of Notch4 (N4) and substantial expression of Notch2 on the surface of -/- cells. Similarly, different TGF- family receptors and considerable levels of p-Smad3 can be detected in these cells, suggesting that these cells are being signaled by TGF-1 in the placental environment. We found that specifically Delta4 had a major impact not only in controlling the immature status of these cells but also in halting the up-regulation of

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CD34 expression on these cells. AlthoughJagged1 was able to maintain these cells in the G0 state, overall TGF-1 had a bigger impact in controlling the cell-cycle progression of these cells by controlling the set of cell cycle regulators. Another important feature observed was that CB -/cells have minimal basal canonical-Wnt activity and are refractory to canonical signals. The combination of a variety of uniquely intrinsic features keeps these cells immature and quiescent and unresponsive to proliferate signals.

PAR-1E-02 MESENCHYMAL STEM CELLS Francesco Lanza,1 Pasini Anna Lisa,1 Diana Campioni2 1

Section of Hematology, University Hospital, Cremona, CREMONA, Italy 2 Section Of Hematology, University of Ferrara, Italy, FERRARA, Italy Although hMSCs (human mesenchymal strem cells) are reported to be uniformly positive for CD90, CD105, CD73, we specifically investigated the hMSC immunophenotype after ex vivo expansion in relation to different parameters such as different culture conditions (serum free, additional use of platelet-lysate, cytokines), culture age, hMSC source (different tissues and normal versus pathologic donors). A multiparametric cytofluorimetric protocol was carried out to distinguish hMSC from the presence of possible endothelial progenitor cells (EPC) growing in mixed culture. Our results further showed that hMSC from amniotic membrane, chorion and BM from patients with hematological malignancies (HM), showed a significant decrease in CD90 (Thy-1) surface molecule expression and elicited a lymphoproliferative allogeneic response in phytohemagglutinin (PHA)/PBMC cultures without any increase in sHLA-G and IL-10 levels. Overall, the results obtained propose a functional role for sHLA-G molecules in inhibiting the PBMC response mediated by MSCs and strengthen the relationship between MSC activation, sHLA-G production and CD90 expression, suggesting the CD90 molecule as a novel predictive marker for hMSC inhibitory ability. Based on these observations, we observed hMSC immunophenotypic modulation, in particular of CD44, CD10, CD146, in relation to the hMSC source and culture passages. The presence of ‘‘contaminant’’ cultured EPC was successfully analysed on CD45neg/CD31pos/CD146pos cultured cells. EPC displayed a variable expression of CD34. These data may be of value in optimising MSC usage in transplantation and regenerative medicine settings.

PAR-1E-03 CIRCULATING ENDOTHELIAL PROGENITOR CELLS Patrizia Mancuso, Francesco Bertolini

European Institute of Oncology, MILAN, Italy Among circulating endothelial cells, two different populations can be discriminated: mature endothelial cell

(CECs) and progenitor cells (CEPs). CECs are the product of the endothelial turnover, have a mature phenotype (Syto 16þ, CD45-, CD31þ, CD146þ) and no proliferative potential when put in culture, whereas CEPs originate from bone marrow, have an immature phenotype (CD45-, CD133þ, CD34þ, VEGFR2þ) and high proliferative potential. CECs and CEPs are increased in different diseases. In particular, in cancer patients, CEPs seem to have a ‘‘catalytic’’ role in cancer growth, progression and recurrence after treatment, while recent clinical data suggest that CEC enumeration might be useful to select and stratify patients whoare candidatefor anti-angiogenic treatments. To use these cells as biomarkers in clinical trials and to compare results from different laboratories, the definition of CEC and CEP phenotype and the standardization of their enumeration are necessary. We developed a flow cytometry technique that unambiguously identify CECs and CEPs. Electron-microscopy and RT-PCR of sorted Syto16þCD45CD31þCD146þ CECs confirmed that these cells were in bona fide endothelial cells by the presence of Weibel-Palade bodies andby the expression ofthe endothelial-specific gene, VE-cadherin. Intra and inter operator assays and test reproducibility were assessed on frozen and fresh samples, and this method was used to detect CECs and CEPs in healthy subjects and cancer patients. In conclusion, our procedure enumerates a truly endothelial cell population with limited intra-reader and interreader variability and can easily be used to investigate other new angiogenic cell populations.

PAR-1S-01 THE ROLE OF LYMPHOCYTES AND MONOCYTES IN STROKES Xabier Urra

Stroke Unit, Hospital Clıńic Barcelona, BARCELONA, Spain The central nervous system (CNS) and the immune system are tightly interconnected. Immunity contributes to the pathogenesis of the ischemic brain injury while CNS lesions can have systemic consequences over immunity. A stroke-induced immunodepression syndrome was described in a murine model of cerebral ischemia, in which animals were at high risk of infectious complications. Infections are also the most frequent complication in stroke patients and could reflect an immunosupressive state after a stroke. In stroke patients, the study of pro- and anti-inflammatory pathways was mainly focused on the assessment of systemic levels of different cytokines. Recently, the study of immune cells has shown rapid changes that are similar in ischemic and hemorrhagic strokes, suggesting that they are mainly an unspecific consequence of the acute lesion of the CNS. Poor prognosis was related to markers of greater inflammation such as high TLR4 expression in monocytes, greater proportion of CD14highCD16- monocytes and greater activation of B cells. The study of the immune cells in patients who had had a stroke also confirmed the exis-



tence of a stroke-induced immunodepression syndrome, characterized by lymphocytopenia and lower TNF- production and low HLA-DR expression in monocytes. Overall, a better understanding of the reciprocal effects between the CNS and the immune system could pave the way to more effective therapies for this devastating condition. These could include strategies aimed at inhibiting Tolllike receptor-4 signaling, promoting minor subpopulations of monocytes that favour tissue repair and angiogenesis and selecting patients at high risk of infection for antibiotic prophylaxis.

PAR-1S-02 CHARACTERIZATION OF REACTIVE OXYGEN SPECIES PRODUCTION (ROS) AND APOPTOSIS IN BLOOD T LYMPHOCYTES FROM PATIENTS WITH CROHN’S DISEASE (CD) Beleń Beltrań,1 Ine´s Moret,2 Francisco Rausell,3 Marıá Garcıá,1 Marisa Iborra,2 Guillermo Bastida,1 Mariam Aguas,1 Elena Cerrillo,1 Pilar Nos,3 Beleń Beltrań3 1

Hospital Unversitario La Fe, VALENCIA, Spain Instituto de Investigacioń Sanitaria del Hospital la Fe, VALENCIA, Spain 3 CIBERehd-Hospital la Fe, VALENCIA, Spain CD patients have increased ROS peripheral leukocyte damage and decreased plasma antioxidants. Aims: To characterise in patient blood lymphocytes at onset of CD disease: Basal and stimulated production of superoxide (O2-), hydrogen peroxide (H2O2) and nitric oxide (NO), their mitochondrial activity, and their apoptotic rates after Fas-treatment. Methods: PBMC were isolated from blood samples of healthy subjects and CD patients by Ficoll. O2-, H2O2 and NO production were measured by flow cytometry. For apoptosis, lymphocytes were cultured for 5 days in the presence of anti-CD3 and anti-CD28. Afterwards, Fas antibody was added and apoptosis was detected by flow-cytometry (Annexin-V) after an ON incubation. Results: Basal and stimulated O2- and H2O2 production are shown in the table. Cm was inhibited in CD patients (8 6 0.5 controls vs 5.3 6 0.3 patients, p< 0.001). The ON exposure to Fas Ab resulted in a significantly less apoptosis in patients than in controls (70.963.1 control vs 56.363.7 patients (p¼0.009). Conclusions: Lymphocytes from CD patients have an increased production of H2O2. The inhibition of m shows impaired mitochondrial activity in CD that facilitates O2-production. Mitochondria inhibition does not depend on NO production. Resistance to apoptosis from CD lymphocytes may be a feature of peripheral cells before reaching the intestinal mucosa. 2

Control CD p-value

[O2]

[O2 þ Pb]

[H2O2]

[H2O2þ Tb]

0.560.1 0.660.1 0.5

3.160.8 1.160.2 0.016

0.560.1 1.160.2 0.03

9.261.3 26.166.9 0.03


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PAR-1S-03 CYTOMICS FOR SAFETY ASSESSMENT IN DRUG DISCOVERY AND REGULATORY TOXICOLOGY Jose-Enrique O’Connor, Guadalupe Herrera, Alicia Martinez-Romero, Laura Diaz, Angela Gomes, Francisco Sala-de-Oyanguren, Julia Platas

Centro de Investigacion Principe Felipe-UVEG, VALENCIA, Spain The prediction of toxicity is relevant for drug safety and. Therefore, regulatory toxicology and test strategies which replace, reduce or refine use of animals (3R) are required. Pharmaceutical companies require early detection of acute or chronic toxicity of a drug candidate. EU policy for Registration, Evaluation and Authorisation of Chemicals implies toxicity assessment for more than 30,000 chemicals. While acute toxicity of a substance or drug candidate is usually detected in vitro, anticipating long-term toxicity is complicated. To predict acute human toxicity, we have miniaturized in vitro cytomic methods based on flow cytometry (FCM) and High-Content Analysis (HCA). To assess their predictive value, the IC50 or EC50 in vitro values for 90 test compounds have been correlated by cluster analysis and hierarchization with reported in vivo LC50 or LD50 in humans and rat. Most assays have good correlation with in vivo human toxicity and classify compounds better than the Global Harmonization System (GHS) based on in vivo rat toxicity. For prediction of chronic hepatotoxicity, we have validated two novel cytomic assays of steatosis and cholestasis, the major causes of drug-candidate withdrawal. Steatosis is approached by an assay consisting of 24-hour exposure of HepG2 cells to fatty acids in the presence of test compounds. Cholestasis is predicted from the interference on bile acid uptake by isolated rat hepatocytes, with a kinetic assay of bile-acid fluorescent derivatives. Our data show that cytomic assays and data mining by array analysis may be very useful in the assessment of drug safety and chemical hazard to humans. Sponsored by EC FP6 (LSHB-CT-2004-512051) and MICINN (BIO2007-65662)

PAR-2E-01 PROGNOSTIC FACTORS IN MASTOCYTOSIS Luis Escribano,1 Ivan Alvarez-Twose,1 Laura Sanchez-Munõz,1 Maria Jara-Acevedo,2 Cristina Teodosio,2 Andres Garcia-Montero,2 Almudena Matito,1 Alberto Orfao2 1

Instituto de Estudios de Mastocitosis, TOLEDO, Spain Servicio de Citometria, SALAMANCA, Spain Mastocytosis comprises a group of heterogeneous diseases characterized by an abnormal expansion and accumulation of mast cells (MC) in different tissues. The clonal nature of mastocytosis can be established through demonstration of gain-of-function mutations involving the tyrosine kinase domain of KIT. Despite the relatively high frequency of signs of biological progression, transformation of ISM to a more aggressive form of the disease was an infrequent event restricted to a

2

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minority of ISM cases; the time from diagnosis to progression varies from 5 to 25 years. Multivariate analysis showed that serum -2 microglobulin levels (p¼0.003), together with the presence of D816V KIT mutation of all hematopoietic lineages (p¼0.02), were the most powerful combinations of independent prognostic factors to predict progression free survival. Regarding overall survival, we have demonstrated that at diagnosis only an age of > 60 years, increased serum alkaline phosphatase, and the development of an associated clonal hematological non-mast cell disorder had independent predictive value for overall survival in ISM. In summary, ISM patients managed with conservative therapy have both a low disease progression rate and long life expectancy. Due to the impact of the presence of D816V KIT mutations in all hematopoietic lineages on disease progression careful follow-up is mandatory; in addition, it points out the relevance of systematically determining, in a clinical setting, the KIT mutational status of both MC and other haematopoietic cells in ISM patients.

PAR-2E-02 THE ROLE OF CD117 MUTATIONS IN MASTOCYTOSIS Andres Garcia-Montero,1 Maria Jara-Acevedo,1 Cristina Teodosio,1 Laura Sanchez-Munõz,2 Ivan A´lvarez-Twose,2 Luis Escribano,2 Alberto Orfao1 1

Centro de Investigacioń del Cańcer/IBMCC (CSIC-USAL), SALAMANCA, Spain 2 Centro Estudios Mastocitosis de Castilla La Mancha, Hospital Virgen del Valle, TOLEDO, Spain Kit (CD117) is a TK transmembrane receptor whose expression has been reported in normal cells [e.g. haematopoietic progenitors, mast cells (MC), Cajal cells, melanocytes, and germ cells]. Activation of Kit by SCF binding plays a major role in haematopoiesis, gametogenesis, MC development and function, melanogenesis and gastrointestinal function. Different KIT mutations can frequently be observed in several neoplastic disorders (e.g. systemic mastocytosis (SM), GIST, seminomas, AML, MDS[3DOTS]), clustering in relatively small regions but most frequently at exon 11 and 17. Interestingly, a clear association between the type of KIT mutation and specific disease groups has been found. In normal MC, activation of Kit signaling through SCF leads to an increased cell proliferation and survival, changes MC migration and adhesion, MC degranulation and mediator release. In SM patients, such effects are typically enhanced by the occurrence of activating KIT mutations, most frequently D816V, which is virtually present in all adults (93%) with Indolent and Aggressive forms of SM. Other KIT mutations were rarely (8 colors at a time) can be pushed forward to measure anything stainable in a cell ¼ Hyperchromatic Cytometry. Different components which



have been suitably combined to achieve this task will be presented. For cell analysis Slide Based Cytometry (SBC) technology is ideal. Unlike flow cytometry, it is non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed after treatment. In this overview the various approaches for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer. The different components which will be discussed are: 1) polychromatic cytometry, 2) iterative restaining (identical fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating colors by different photostability), 4) photoactivation (of fluorescent nanoparticles or photocaged dyes), and 5) photodestruction. Based on the relocation feature immobilized cells on a microscope slide can subsequently be reanalyzed and the data collected for the identical cells and information merged on the single cell level after manipulation steps. With intelligent combination of several different techniques. hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on individual cells. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

PAR-2S-02 POLYCHROMATIC FLOW CYTOMETRY FOR STUDYING IMMUNE DEFICIENCIES Andrea Cossarizza

Univ. of Modena and Reggio Emilia, MODENA, Italy The use of polychromatic flow cytometry (PFC) is playing a crucial role in understanding the behaviour of the immune system. In the last years, my group has analyzed different conditions characterized by severe immune deficiency, such as the infection by the human immunodeficiency virus (HIV) in patients who had received an organ transplantation and had immunosuppressive therapy. Polyfunctional (i.e.,capable of more than 2 activities) CD8þ T cells specific for HIV play a role in controlling the production of the virus, and thus in delaying the progression of the infection. Less is known about the role of such cells and of T regulatory cells (Treg) in ‘‘long-term non-progressors’’ (LTNP), i.e. those patients infected from at least 10 years, with a stable number of CD4þ T cells (>500 CD4þT cells/uL), and who never had antiretroviral therapy. We analyzed 10 LTNP, 8 treatment-naı¨ve HIVþ patients with progressive disease and 8 patients who underwent CD4-guided Structured Treatment Interruption and had to restart therapy. By PFC, using a 16 parameters CyFlow ML (Partec, Germany), we evaluated HLA-DRþ/- Treg (CD3þ,CD4þ, CD25þ,FoxP3þ,CD127-), and detected HIV-specific CD4þ and CD8þ T cells by simultaneously evaluating the expression of CD107a, CD154, IL-2, IFN-gamma after stimulation with Gag overlapping peptides and exclusion of dead cells. A similar study was performed in patients undergoing organ transplantation, where we compared the effect of two immunosuppressive protocols with Cyclosporine A (CsA) or


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Everolimus (Evr). Data showing the utility of PFC in the clinical practice will be presented and discussed.

PAR-2S-03 PREDICTION OF CHRONIC LIVER DAMAGE BY HIGH-CONTENT CYTOMETRY Sandra Pinto,1 Alicia Martinez-Romero,1 Laura Dıáz,1 Maria Jose´ Go´mez-Lechoń,2 Jose´ Vicente Castell,3 Jose´ Enrique O’Connor1 1

Laboratory of Cytomics, Mixed Unit CIPF-UVEG, VALENCIA, Spain 2 Laboratory of Experimental Hepatology, Research Foundation of Hospital La Fe, VALENCIA, Spain 3 University of Valencia, VALENCIA, Spain Multiparametric analysis of xenobiotic toxicity with cellular-based technologies such as high content assays (HCA), are of great importance for the detection of toxicity and the classification of compounds based on observed patterns of cellular injury. Furthermore, by analysing more than one cell parameter at a time, they offer the opportunity to study cellular events and processes in their true biological context,and uncover relationships between them,. In this work, we present the HCA we developed for predicting chronic liver damage. The assays were performed on rat hepatocytes and in HepG2 cell cultures to detect and analyse the effect of xenobiotics on liver steatosis (i.e. neutral lipid accumulation) and phospholipidosis (i.e. phospholipid accumulation). To achieve this, we used BODIPY1 (4,4-difluoro3a,4a-diaza-s-indacene) as tracer for nonpolar lipids, as well as the high-content screening kit LipidTox (Molecular Probes) which detects both steatosis and phospholipidosis. Moreover, we evaluated changes on mitochondrial membrane potential using Tetramethylrhodamine (TMRM). The HCA were performed in the automated cell imager IN Cell Analyzer 1000 (GE HealthCare) using suitable excitation and emission filters, and dichroics. Thereafter, the IN Cell Investigator software packages (GE HealthCare), were used with appropriate algorithms for cell segmentation and data calculation. We conclude that HCA provide complementary and consistent results with our Flow Cytometry assays,previously developed. In addition, HCA reveal cell heterogeneity and may provide tools to address this phenomenon. Sponsored by EC Project Predictomics (LSHB-CT-2004512051) and GE Healthcare.

PAR-3E-01 NKT CELLS IN HYPERSENSITIVITY PNEUMONITIS AND SARCOIDOSIS Peter Korosec

University Clinic of Respiratory and Allergic Diseases, GOLNIK, Slovenia NKT cells comprise a unique subgroup of lymphocytes that express features of both T and natural killer (NK) cells. These cells co-express T cell receptors (TCR) as well as markers associated with NK cells, such as CD56 and/or

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CD161. In humans, two major subsets of natural killer T cells have been described. The more widely studied subset are the invariant NKT cells (or type I), which express a highly restricted T cell receptor with an invariant V24J18 chain, preferably paired with V11, and are dependent on the presentation of the glycolipid antigen through CD1d, a member of class I non-polymorphic antigen presenting molecules. The second class of NKT cells (or type II), CD1d- independent cells, expresses a TCR/ repertoire and is independent of CD1d for their activity. We recently demonstrated the expansion of pulmonary NKT cells in patients with hypersensitivity pneumonitis. The major NKT phenotype in hypersensitivity pneumonitis was non-biased CD3þCD8þCD56þ. Furthermore, NKT cells might contribute to the amplified and prolonged CD4-positive Th1 response that characterizes sarcoidosis. We showed significant reduction of invariant NKT cells both in the lung and in peripheral blood of patients with sarcoidosis. Moreover, we demonstrated that this deficiency was associated with changes in SLAM/SAP signaling, which is a major pathway involved in invariant NKT cell development. Finally, we recently closely followed the progress of invariant NKT cells and SLAM/SAP signaling in the peripheral blood of patients with sarcoidosis and in control subjects over a long period.

PAR-3E-02 T CELL ACTIVATION IN EXACERBATIONS OF REFRACTORY ASTHMA Antoine Magnan,1 Karine Botturi-Cavaille`s2 1

L’Institut du Thorax, NANTES, France INSERM, NANTES, France Refractory asthma is an ultimate form of asthma defined by a high frequency of exacerbations in patients already treated with high doses of inhaled steroids, long acting beta-2 agonists and frequently oral corticosteroids. Severe exacerbations represent the main issue in refractory asthma management, inducing heavy costs, leading to emergency care requirement and, all too often, intensive care treatment and ultimately, death. It is therefore necessary to dispose of valid early markers of exacerbations, robust enough to intensify the asthma treatment before symptoms. Asthma results from an inflammatory reaction mainly driven by Th2 cells and eosinophils. Exacerbations are triggered by allergens, pollutants and viruses. Induced sputum is an easy and safe procedure to collect bronchial cells and can be repeated in the same subjects longitudinally. We performed a study in induced sputum from refractory asthmatics to detect whether a change in T cell cytokine production could predict an exacerbation. Samples were obtained in stable state before, during and after 16 exacerbations in 30 patients. Blood samples were taken concomitantly. IFN-g, IL-4, IL-5, IL-13 T cell production was detected by flow cytometry in T cells and in the CD8þ subset. A mixed Th1/Th2 activation was detected before exacerba-

2

tions that lasted during exacerbations but returned to baseline thereafter. A similar profile was observed in blood and induced sputum. A new study is in process to validate the blood T cell activation as a biomarker, taking viral infections into account. This study was sponsored by the Programme Hospitalier de Recherche Clinique, INSERM, and Re´gion Pays de La Loire.

PAR-3E-03 CELLULAR IMMUNITY AGAINST TB IN BLOOD AND BRONCHOALVEOLAR LAVAGE FLUID Simon Barry

University Hospital LLandough, CARDIFF, United Kingdom In the majority of cases tuberculosis (TB) is controlled by the human host and only approximately 10% of asymptomatic infected individuals develop active TB disease over their lifetime. The fact that HIV co-infection, which selectively depletes CD4þ T cells, increases this chance of reactivation from 10% per lifetime to 10% per annum emphasises the role of immunoregulation of TB. Furthermore, studies of knockout mice that lack the capacity to generate type-I cytokine responses, together with rare cases of humans lacking IL-12 and IFN-g receptors, have highlighted the most important mechanisms involved in immune control. It is well appreciated that mycobacterium tuberculosis is usually acquired by inhalation and results in predominantly respiratory disease. It follows that anti-TB defences will concentrate at the site of infection and that the focus of investigation should be lung based. Flow cytometry, investigating functional lung T lymphocyte responses following short term incubation with TB antigens, is the optimum tool for studying the immune response to TB. It has been demonstrated that lung CD4þ T lymphocyte IFN- and TNF- responses following incubation with purified protein derivative (PPD) were 300 fold greater than in the blood. A dominant lung CD4þ T lymphocyte response is also seen in non-pulmonary TB. Interestingly, prior BCG vaccination does not influence the lung responses to PPD, unlike in the blood where the discriminatory RD-1 antigens, ESAT-6 and CFP-10 must be used. In subjects with advanced, cavitary lung disease, there is a waning of the lung lymphocyte responses and an accompanying neutrophilia, perhaps indicative of a loss of immune control.

PAR-3S-01 DETECTION OF CIRCULATING TUMOR CELLS IN HUMAN BREAST CANCER Marıá Campos, Jose´ Juan Gaforio, Fernando Warleta, Cristina Sańchez-Quesada

University of Jaeń, JAEŃ, Spain Breast cancer (BC) is the most frequent type of cancer and the third highest cause of cancer death in European women because of its high incidence and good prognosis.



Frequently, death results from the development of metastases. BC has been shown to shed tumor cells into the circulation, even at the earliest stages of primary tumor development, and these cells have been isolated in peripheral blood (PB) and in bone marrow. Pretlow et al. (2000) have demonstrated that Circulating Tumor Cells (CTCs) isolated from PB of cancer patients are able to develop metastasis. Thus, the early detection of CTCs may have important therapeutic and prognostic implications. Nowadays, it has been demonstrated that bone marrow tumor cell detection in BC is a known prognostic factor for disease-free and overall survival, and the detection of CTCs in PB has clinical relevance, not just as an indicator of overall survival, but also as disease progression, as confirmed by our group. In short, our results showed that CTCs cells in PB were identified in 62% of BC patients. There were significant differences in the presence of CTCs according to estrogen receptor expression (p¼0.049), and lymph node status (p¼0.033), but not to other characteristics. The median follow-up was 21 months and the detection of CTCs, before starting the chemotherapy, in these patients was significantly correlated with progression-free survival (p¼0.058) and overall survival (p¼0.003). Recently, we published a novel method enabling simultaneous immunophenotyping and genetic analyses of CTCs. This could be useful for a better classification of patients according to genetic features of CTCs and for the application of specific therapies.

PAR-3S-02 MINIMAL RESIDUAL DISEASE IN HEMATOLOGICAL MALIGNANCIES Neus Villamor

Unitat d’Hematopatologia, Hospital Clinic de Barcelona, BARCELONA, Spain Multiparametric flow cytometry (MFC) plays a major role in the diagnosis of hematological malignancies and is becoming more important in the evaluation of the response. The persistence/reappearance of submicroscopic leukemic cells is an important post-treatment prognostic factor for the outcome of patients with acute leukemia (AL), chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). The MFC assay for MRD is generally less sensitive than molecular techniques, but it is also less complex. The detection of leukemic associated phenotype (LAIP) is the basisof MRD assay. The use of 4 antigenic combinations with core and specific markers allows a sensitivity of 0.01% abnormal cells in the majority of acute lymphoblastic leukemia, CLL and MM patients. This sensibility is only obtained in half the cases with acute myeloblastic leukemia. The most important source of false positive and negative results in the MRD assay in patients with AL is the presence of leukemic subpopulations at diagnosis, changes induced by therapy and phenotypic shifts during follow-up. Although MRD results are used to stratify patients with AL in post-induction therapy, the clinically relevant time-points


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and cut-off values have not been clearly established. In CLL or MM, a MRD test reaching a sensitivity of at least 0.01% is required to consider a patient to be in CR MRD negative. In hematological diseases, the criteria of CR is moving from morphology to MRD, and the challenge in the future is the standardization of methodology and terminology for MRD detection in each type of disease and to provide a common base for clinicians to compare results between different clinical trials.

PAR-3S-03 POLYCHROMATIC FLOW CYTOMETRY STUDIES OF DENDRITIC CELLS AND MONOCYTES IN AUTOINMUNE DISEASES. Jorge Monserrat,1 Miguel Angel Sanchez,2 Ana Sanchez Atrio,3 Ana Perez,3 Maria Jose Leon,3 Luis Chara,2 David Diaz,2 Hugo Barcenilla,2 Zaida Moreno,2 Alfredo Prieto,2 Eduardo Reyes,2 Melchor Alvarez-Mon3 1

Universidad de Alcala, ALCALA DE HENARES, Spain Lab. de enfermedades del S. Inmune. Dto. Medicina. F. Medicina. UAH. CSIC/IMMPA, ALCALA´ DE HENARES, Spain 3 Servicio de enfermedades del Sistema Inmune (ESI). Hospital Principe de Asturias, ALCALA´ DE HENARES, Spain Dendritic cell (DCs) and monocyte studies have been demonstrated to be important for the compression of the different mechanisms in human pathologies. Newer markers appear in peripheral blood which study their distribution and activation stage. Objective: By using polychromatic flow cytometry in 11 colors, we have studied the distribution and activation subsets of monocytes and DCs in peripheral blood from autoimmune diseases patients with or without biological treatments (anti-TNF). Methods: We have studied peripheral blood monocytes and DCs from patients with rheumatoid arthritis (RA), ´ s Disease and healthy controls. For surface labeling Crohn antibodies against the antigens on monocytes we used: CD(3,56,19,14,16,62L,11b), Slan, HLA-DR, CX3CR1 and dead cells exclusion probe; and on DCs: CD(3,56,19,14,16,11c,1c,62L,11b), HLA-DR, CX3CR1 and dead cells probe. We acquired in a FacsAria. Results: We found significant differences in the number of plasmacytoid and myeloid I cells that were decreased in RA and Crohn patients with regard to healthy controls. After 12 months of anti-TNF treatment, the levels were normalized in both groups of patients. Myeloid cells and plasmacytoid cells showed a significant decrease in the CD62LþCX3CR1þ and CD11bþCD62Lþ expression respectively, with a significant increase after 12 months of treatment. In monocytes, we found that RA and Crohn patients showed a decrease of classical monocytes defined CD14BrþCD16- and showed an increase of transition subsets defined by CD14BrþCD16Br/Lwþ. Conclusions: Anti-TNF treatment is able to normalize the distribution and activation stage of peripheral blood monocytes and DCs in RA and Crohn patients. 2

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PAR-4E-01 ACUTE AND CHRONIC NK-CELL ACTIVATION Margarida Lima

Hospital de Santo Antońio, Centro Hospitalar do Porto, PORTO, Portugal NK-cell (NKC) activation is tightly regulated through signals that come in a variety of forms, including a complex repertoire of receptors/ligands, as well as soluble factors. Activated NK-cells express a number of molecules that are absent in resting NKC; at the same time, they down-regulate or upregulate receptors that are constitutively expressed. Our experience on the immunophenotypic analysis of NKC in normal individuals, patients (pts) with acute viral infections (AVI), chronic infections (CI) or tumors (T) allowed us to define early and late NKC activation-related phenotypic patterns. Normal CD56þlow NKC: the majority of CD56þlow NKC present in normal blood are CD2-/þlow, CD7þhigh. Nearly all cells are CD11bþ, CD38þ, HLA-DR- and CD45ROand express high levels of CD11a and CD45RA. In contrast, CD11c and CD57 are expressed in a variable fraction of cells. Recently activated CD56þlow NKC: CD56þlow NKC from pts with AVI have a pattern of CD2 and CD7 expression similar to that observed in normal CD56þlow NKC, except that of a higher % of CD2þ cells. They are typically CD38þhigh and CD11bþlow, express dimly and heterogeneously CD11c and only a few are CD57þ. In addition, most of them are HLA-DRþ and CD45R0 is transiently expressed in a variable fraction of cells. Chronically activated CD56þlow NKC: CD56þlow NKC from pts with CI or T are typically CD2-/þhigh, CD7þlow. Simultaneously, they exhibit a CD38-/þlow CD11b-/þlow phenotype and express high levels of CD57, while CD11c expression is dim and heterogeneous. At this stage, most NKC have already reverted into their original HLA-DR- CD45RO- phenotype, although variable proportions of HLA-DRþ and/or CD45ROþ are found.

PAR-4E-02 HOW NK CELLS INFLUENCE DISEASE Loris Zamai

University of Urbino, URBINO, Italy Natural Killer (NK) cells kill HLA class I (HLA-I) negative virus-infected and tumor cells utilizing both activatory and inhibitory receptors. Information available in the field of NK cell receptors, focusing on their role in cell-mediated immune response, will be presented. Among NK receptors, HLA-I-binding inhibitory receptors, known to prevent NK cell cytotoxicity against itself, have been shown to be crucial in ‘‘licensing’’ NK cell to kill HLA-I negative transformed cells. These receptors include the - evolutionarily recent killer immunoglobulin-like receptors (KIRs) that, most probably, are specialized to fight newly evolved diseases. These receptors recognize oligomorphic regions of HLA Class I molecules (KIR-ligand). In fact, KIR ligand-mismatch regimens have proven the efficacy of NK cell-based immuno-

therapy and, recent epidemiological data, indicate that some allelic variants of KIRs expressed on NK cells protect against viral-infections and cancer. Emerging evidences on NK cell biology will be exploited in an effort to explain how distinct alleles of KIRs could influence infections and tumor progression.

PAR-4E-03 CHARACTERIZATION OF THE HUMAN NK CELL RESPONSE TO CYTOMEGALOVIRUS-INFECTED MONOCYTE-DERIVED DENDRITIC CELLS Miguel Lo¨pez-Botet,1 Giuliana Magri,2 Aura Muntasell,1 Neus Romo,2 Diogo Baia,2 Medya Shikhagaie2 1

University Pompeu Fabra. IMIM-Hospital del Mar, BARCELONA, Spain 2 University Pompeu Fabra, BARCELONA, Spain Myeloid dendritic cells play a central role in the biology of human cytomegalovirus (HCMV) infection as they differentiate from myeloid progenitors, which constitute a main site for viral latency, and are themselves susceptible to direct infection. NK cells are involved in immune defence against HCMV though information on the nature of NK cell receptors involved is limited. In the present study, we analysed the response of NK cell populations against autologous monocyte-derived dendritic cells (moDC) infected by the TB40/E HCMV strain, assessing the antagonistic effect of monoclonal antibodies (mAbs) specific for different activating NK cell receptors (NKp46, NKp30, NKG2D, DNAM1) and the expression of their ligands. Interaction with autologous HCMV-infected moDC, which down-regulated the expression of HLA class I and II molecules, specifically activated NK cells, triggering IFN production and cytotoxicity. Only NKp46 and DNAM-1-specific mAbs inhibited NK cell activation in response to HCMVinfected moDC, despite the fact that their ligands were partially down-regulated in infected cells. Differences in the efficiency of NK cell subsets bearing the inhibitory CD94/ NKG2A or activating CD94/NKG2C NKR specific for HLA-E to respond against infected moDC were noticed. Remarkably, cytokines with a key role in the anti-viral immune response (i.e. type I IFN and IL-12) selectively regulated the expression of some NKR (i.e. NKG2A and NKG2D). Our results indicate that NK cells may efficiently respond to HCMV-infected immature moDC, overcoming putative viral immune evasion strategies..

PAR-4S-01 CHARACTERIZATION OF A SIDE POPULATION OF CANCER CELLS FROM HUMAN BRAIN TUMORS Jordi Petriz,1 Jana Balbuena,2 Purroy Noelia,1 Castresana Javier S.2 1

Hospital Universitari Vall d’Hebron - Institut de Recerca (VHIR), BARCELONA, Spain 2 Brain Tumor Biology Unit-CIFA, PAMPLONA, Spain The expression of ABC transporters has been classically associated with the phenomenon of multidrug resistance in



cancer cells. Conceivably, prevention of ABC transporter induction in cancer cells might help to avert drug resistance. ABC proteins are involved in the transport of numerous endogenous substrates and play an important role in protecting tissues against the penetration of toxins across the blood-brain barrier, the blood-cerebrospinal-fluid barrier or the endothelium. Therefore, prevention of transporter induction in cancer might increase the undesired penetration of toxins in non-cancer normal cells. ABCB1, ABCC1 and ABCG2 can confer the multidrug resistant phenotype to tumor cells in vitro and have a broad spectrum of binding for generally hydrophobic substrates. ABCB1 and ABCG2 transporters are respectively highly conserved in normal CD34-positive and CD34-negative stem cells and may play a role, not only in protecting these cells from cytotoxic agents, but also from endogenous or physiological substrates. ABCG2 confers the Side Population phenotype and enhances hypoxic cell survival, having a key role in hematopoietic stem cells concentrated in hypoxic areas. Interestingly, enforced expression of ABCG2 inhibits hematopoietic development and it has been proposed as a regulator in extracellular signals that influence stem cell interactions with the microenvironment. Possible roles of ABC transporters expressed in normal cells have been suggested by disruption of their function. In this study, we show that ABCG2 is orchestrating gene expression programs by means of an active efflux of substrates involved in intercellular and/or intracellular cell signaling.

PAR-4S-02 HUMAN ENDOMETRIAL SP EXHIBIT GENOTYPIC, PHENOTYPIC AND FUNCTIONAL FEATURES OF SOMATIC STEM CELLS Irene Cervello´

Fundacioń IVI-Instituto Universitario IVI, Universidad de Valencia, VALENCIA, Spain During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis of a woman’s cycle and its dysfunction may be involved in the etiology of pathological disorders. Somatic stem cells (SSC) are defined as a restricted subpopulation of quiescent, slow-cycling, undifferentiated resident cells, characterized by a high proliferative capacity, multipotentiality, capability of self-renewal and the ability to form the tissue from which they originate. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cell population. The existence of endometrial SSC has been postulated, and different groups, using different approaches, have demonstrated the existence of SSC-like cells in the human and murine endometrium.. Our group explores the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation


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capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium following subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

PAR-4S-03 SIGNALING THROUGH TLR2/MYD88 INDUCES DIFFERENTIATION OF MURINE HEMATOPOIETIC STEM AND PROGENITOR CELLS TO FUNCTIONAL PHAGOCYTES IN RESPONSE TO CANDIDA ALBICANS Marıá Luisa Gil,1 Alberto Yań˜ez,1 Celia Murciano,1 Daniel Gozalbo,1 Jose´ Enrique O’Connor2 1

Universitat de Vale`ncia, VALENCIA, Spain Centro de Investigacioń Prıńcipe Felipe, VALENCIA, Spain Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells. Therefore, these receptors may play a role in hematopoiesis in response to pathogens during infection. It is well established that TLRs, mainly TLR2 and TLR4, are involved in the host interaction with Candida albicans; hence, we studied for the first time, the interaction of a microorganism, such as C. albicans, with murine hematopoietic stem and progenitor cells (from C57BL/6, MyD88-/-, TLR2-/- and TLR4-/- mice) in defined in vitro culture conditions. Results showed that inactivated yeasts and hyphae of C. albicans induce in vitro the proliferation of murine haematopoietic stem and progenitor cells (LKS: Lin- c-Kitþ Sca1þ) as well as their differentiation to lineage positive cells, through a MyD88-dependent pathway. This process is mainly mediated by TLR2, and expanding cells express myeloid (CD11b) and not lymphoid (B220) markers. In addition, this signaling promotes the differentiation of common myeloid progenitors (CMPs) and granulocyte and macrophage progenitors (GMPs) into cells with the morphology of macrophages and neutrophils, characterized by an increase in the expression of CD11b, F4/80 and Ly6G, independently of the presence of growth and differentiation factors. These in vitro differentiated cells were able to phagocytose C. albicans yeasts and to produce proinflammatory cytokines. In conclusion, C. albicans may be sensed by TLRs on hematopoietic stem and progenitor cells to promote the host capability for rapidly replenishing functional mature myeloid cells that constitute the first line of defence against C. albicans. 2

POS-FU-01 EP4 PROSTANOID RECEPTOR SIGNALING IN HUMAN EOSINOPHILS Eva Sturm, Gerald-Peter Parzmair, Petra Luschnig-Schratl, Viktoria Konya, Akos Heinemann

Medical University of Graz/Institute of Experimental and Clinical Pharmacology, GRAZ, Austria Background: The accumulation of eosinophils in lung tissue is a hallmark of asthma and it is believed that eosino-

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phils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and broncho-protective mechanisms in asthma, but the underlying mechanisms have not been fully explored. Therefore, the aim of this study was to investigate the role of the PGE2 receptor EP4 in eosinophil effector function. Methods: Eosinophil shape change and Ca2þ flux responses were measured in human PMNL samples using flow cytometry. For chemotaxis studies, human eosinophils were purified by negative magnetic selection; chemotaxis was determined in 48-well microBoyden chambers and migrated eosinophils were enumerated by flow cytometric counting. Results: The selective EP4 agonist ONO-AE1-329 dosedependently attenuated eosinophil function. The inhibition of eotaxin-induced chemotaxis was prevented by the selective protein kinase inhibitors LY-294002, chelerythrine, U0126, triciribine and BX-912. The ONO-AE1-329 induced decrease of eosinophil shape change responses seemed to be associated with PKC/PDK1/AKT but not with PI3K activation, and EP4-mediated inhibition of eosinophil Ca2þ flux could only be prevented by the PDK1 inhibitor BX-912. Conclusion: Our findings indicate that the selective EP4 agonist ONO-AE1-329 inhibits eosinophil chemotaxis, shapes change and Ca2þ flux via three distinct but PDK1-activating pathways. These data support the concept that selective EP4 receptor agonists may represent a new class of drugs useful for the treatment of eosinophilic diseases because of their direct action on eosinophil effector function.

POS-FU-02 LABORATORY DETECTION OF HEPARIN-INDUCED TROMBOCYTOPENIA: FUNCTIONAL ASSAY BY FLOW CYTOMETRY Elvira Malicev, Tomaz Jerlah, Tadeja Dovc-Drnovsek, Primoz Rozman

Blood Transfusion Centre, LJUBLJANA, Slovenia Heparin sometimes causes trombocytopenia and concomitant activation of coagulation cascade which could result in arterial or venous thrombosis. Diagnosis of heparin-induced trombocytopenia (HIT) should be confirmed by laboratory testing in all patients suspected of having HIT. Antigen-binding assays (ELISA) are commercially available and are commonly used for detecting the presence of heparin-dependent platelet antibodies in serum. Although the sensitivity of the ELISA assay is very high, the specificity of this assay is relatively low. Because not all detected antibodies are capable of activating platelets, it is recommended that further functional assay be performed when sera tests positive in the ELISA assay. Functional assays detect heparin-dependent platelet activation as a result of exposure of normal platelets to patient serum. They are technically challenging as tested platelets can readily be activated by in vitro manipulations. To lower platelet activation, an in-house method was developed by detecting heparin-dependent platelet activation in whole blood. Our study, which included 78 patients referred for laboratory diagnosis of HIT, showed that 50 sera were positive

and 28 sera were negative in ELISA IgG assay. Functional assay by flow cytometry found out that only 26 of ELISA positive and 1 of ELISA negative sera were able to induce donor platelet activation. This preliminary study indicates that 48% of patients with detected heparin-dependent antibodies are unlikely to activate platelets and confirms previous reports that only a minority of sera reactive in immunoassays are capable of causing HIT.

POS-FU-03 STUDY OF MITOCHONDRIAL MEMBRANE POTENTIAL IN HEPG2 BY COMPARISON BETWEEN LASER SCANNING CYTOMETRY AND CYAN-HYPERCYT FLOW CYTOMETRY Ana Juan-Garcia,1 J.Paul Robinson,2 V. Jo Davisson,3 Cristina Juan,4 Guillermina Font4 1

Purdue University Cytometry Laboratories, WEST LAFAYETTE, United States of America 2 PUCL Dept. of Basic Medical Sciences and Dept. of Biomedical Engineering, WEST LAFAYETTE, IN, United States of America 3 Dpt. of Medicinal Chemistry and Molecular Pharmacology Purdue University, WEST LAFAYETTE, IN, United States of America 4 Dept. de Ciencies de l’Alimentcio i Toxicologia, Universitat de Valencia, BURJASSOT-VALENCIA, Spain Laser Scanning Cytometry (LSC) is a powerful tool for qualitative and quantitative analysis of cell populations in situ in preclinical drug development. On the other hand, CyAn-HyperCyt high throughput flow cytometry is designed for speed, efficiency and versatility of suspended cells. Peroxisome Proliferator-Activated Receptor is a type of nuclear regulatory protein involved in transcription of genes regulating glucose and fat metabolism. It plays an essential role in the regulation of cellular differentiation, development and metabolism. Different compounds interfere in its tasks and give rise to a dysfunction in mitochondrial membrane potential (MMP). Five hypoglycemic compounds, three thiazolidinediones and two biguanides,and six lipid modifiying compounds, three fibrates and three statins have been analyzed in HepG2 cells with high content toxicity screening and the comparison between techniques, LSC and CyAn-HyperCyt flow cytometer is presented. High-content data were obtained using a set of four fluorescent biomarkers (TMRM, Hoechst 33342, ToPro-3, DCFDA), enabling characterization of MMP, plasma membrane permeability, reactive oxygen species generation, DNA content, nuclear circularity and area. Cell cycle analysis was performed with propidium iodide by FC and confirmed by lactate dehydrogenase assay. A summary of these data is presented. An altered mitochondrial response in HepG2 cells was observed after prolonged incubation (24 h). A decline of MMP was accompanied by decreasing cell viability and changes in the cell cycle. Acknowledgements: A.J.G. thanks the Spanish MCINN for the Postdoctoral Grant (MICINN/Fulbright, EX20080452). Plan Nacional I-DþI (2008-2011)



POS-FU-04 THE ASSESSMENT OF A549 LUNG CANCER CELLS RESPONSE TO CURCUMIN CONSIDERING CYTOSKELETAL ORGANIZATION Mariusz Andrzej Szczepanski,1 Lidia Gackowska,2 Izabela Kubiszewska,2 Anna Litwiniec,1 Dariusz Grzanka,3 Alina Grzanka1 1

Depart. of Histology and Embryology, Collegium Medicum, N. Copernicus University, BYDGOSZCZ, Poland 2 Department of Immunology, Collegium Medicum, N. Copernicus University, BYDGOSZCZ, Poland 3 Depart. of Clinical Pathomorphology, Collegium Medicum, N. Copernicus University, BYDGOSZCZ, Poland The cytoskeleton of eukaryotic cells structure plays a crucial role in most processes of living cells. Here we wished to elucidate the effect of curcumin treatment on A549 cell line, including MF and MT cytoskeletons changes. A549 cells were treated with appropriate concentrations of curcumin (0-360 M curcumin) for 24, 48 and 72 hours. At indicated time points, the following experimental procedures were performed: flow cytometry procedures: intracellular staining of F-actin and -tubulin, double-staining with Annexin V and 7-AAD, TUNEL assay, PI/RNAse cell cycle analysis; cellular morphology: immunofluorescence of F-actin and -tubulin; conventional TEM and colorimetric MTT assay. Our results show that curcumin inhibits the viability of A549 in a dose- and time dependent manner. Cell death processes were associated with cellular shrinkage, disorganization of the architecture of MF and MT cytoskeletons and a decrease in the intracellular levels of F-actin and -tubulin. Annexin V staining confirmed the increase in the percentage of apoptotic and necrotic cells. Moreover, cell cycle analysis revealed the increase in the percentage of sub-G1 and G2/M cells after curcumin treatment; however, a TUNEL assay has not confirmed the DNA fragmentation. Our results suggest that curcumin treatment leads to changes in MFs and MTs that may mediate various cellular responses, such as cell survival and cell death, depending on the curcumin concentration. The mode of cell death induced by curcumin is thought to be mainly apoptotic-like with lower concentrations and necrotic after treatment with higher concentrations of the agent.

POS-FU-05 PULSED ELECTROMAGNETIC FIELDS (PEMFS) INSTIGATE CALCIUM-DEPENDENT STEM CELL ACTIVATION OF MECHANOSENSITIVE TISSUES Malgorzata Kisielow,1 Jack Traxler,2 Christian Beyer,3 Juerg Froelich,3 Alfredo Franco-Obregon4 1

University and ETH Zurich/Institute for Biomedical Engineering, ZURICH, Switzerland 2 Swiss Federal Institue of Technology - ETH Zurich, ZURICH, Switzerland 3 Lab. for Electromagnetic Fields and Microwave Electronics, ETH Zurich, ZURICH, Switzerland 4 Institute for Biomedical Engineering, ETH Zurich, ZURICH, Switzerland


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Pulsed electromagnetic fields (PEMF) have been clinically shown to promote the healing of chronic bone fractures. However, the molecular mechanism of this regenerative effect is unclear. One common intracellular mediator associated with PEMF treatment, in isolated cells as well as in vivo, is calcium. We therefore examined Ca2þ-mediated cellular pathways in mechnosensitive tissues that are responsive to PEMFs. Mouse C2C12 skeletal muscle cells possess stretch-activated Ca2þ channels (SACs) similar to those expressed in bone. We thus examined the effect of PEMF on C2C12 myoblasts. Our initial experiments have shown that 10 min episodes of PEMF greatly augment ( by 80%) the proliferation of myoblasts after only 15 hours. Importantly, this increase in cell number is inhibited by agents impeding Ca2þ influx through SACs, indicating that PEMFs open SACs, thereby initiating myoblast expansion. Ca2þ activates the transcription of muscle-specific genes via NFAT. One target of NFAT is the IGF-1gene that is essential for the development of all mechanosensitive tissues. In skeletal muscle, the IGF-1 gene is alternatively spliced, depending on mechanical stimuli. Dynamic mechanical stimuli promote the expression of the IGF-1Eb isoform which is then responsible for activation of the myogenic satellite pool. To test the connection between mechanically triggered Ca2þ entry and myoblast activation, we employed NFAT responsive- EGFP reporter system in C2C12 cells. This approach allows for easy monitoring of NFAT activation, as well as isolation and analysis of activated cells.

POS-FU-06 FUNCTIONAL CHARACTERIZATION OF PERIPHERAL BLOOD DENDRITIC CELLS AND MONOCYTES IN SYSTEMIC LUPUS ERYTHEMATOSUS Tiago Carvalheiro,1 Ana Henriques,1 Luı´s Ineˆs,2 Isabel Silva,1 Maria de Jesus Inaćio,1 Susana Pedreiro,1 Maria Luı´sa Pais,1 Jose´ Antońio Pereira da Silva,2 Artur Paiva1 1

Centro de Histocompatibilidade do Centro, COIMBRA, Portugal 2 Serviço de Reumatologia dos Hospitais da Universidade de Coimbra, COIMBRA, Portugal Systemic Lupus Erythematosus (SLE) presents numerous immunological and clinical manifestations, characterized by a large amount of circulating autoantibodies as a result of abnormal activation of autoreactive B and T helper cells, probably as a result of defects in antigen presenting cells (APC) function. Our aim was to evaluate the frequency and functional ability of monocytes and dendritic cells (DC), namely myeloid (mDC) and CD14-/lowCD16þ DC subsets as well as produce pro-inflammatory cytokines (TNF-, IL-1, IL-6 and IL12) in peripheral blood (PB) from SLE patients. We studied 34 SLE patients divided into active (SLEDAI 5) disease (AD; n¼15) and inactive (SLEDAI 70 years confirmed the presence of - frequently multiclonal - CLL-like B-cells in all but one of them. Overall, these results support the theory that over a certain age, the emergence of one or more CLL-like Bcell populations would occur. Further studies are required to elucidate whether these cells represent the physiological counterpart of CLL vs an ancestral leukemic cell.

POS-CC-09* PREVALENCE AND PHENOTYPIC/GENETIC CHARACTERISTICS OF ATYPICAL NON-CLL-LIKE MBL (MONOCLONAL B-CELL LYMPHOCYTOSIS) Wendy Nieto,1 Cristina Teodosio,1 Antonio Lo´pez,2 Ma Aranzazu Rodrı´guez-Caballero,1 Alfonso Romero,3 Paloma Ba´rcena,2 Ma Laura Gutierrez,2 Anton W Langerak,4 Paulino Fernandez-Navarro,5 Alberto Orfao,1 Julia Almeida,1 And the Primary Health Care Group of Salamanca6 1

Centro de Investigacioń del Cańcer, SALAMANCA, Spain Servicio de Citometrıá y Departamento de Medicina Universidad de Salamanca, SALAMANCA, Spain 3 Gerencia de Atencioń Primaria de Salud de Salamanca, Sanidad de Castilla y Leoń, SALAMANCA, Spain 4 Of Immunology, Erasmus MC, Rotterdam, ROTTERDAM, Netherlands 5 Centro de Atencioń Primaria de Salud de Ledesma, Salamanca, Sanidad de Castilla, SALAMANCA, Spain 6 Spain Monoclonal B-cell lymphocytosis (MBL) indicates 40 years old (62613years) with normal lymphocyte counts were immunophenotyped using high-sensitive flow cytometry, based on 8-color stainings and screening for >5x106 total PB leukocytes. Thirteen subjects (2.0%; 9 males/4 females, aged 73610years; absolute lymphocyte count: 2.460.8x109/L) showed a non-CLL-like clonal B-cell population, whose frequency clearly increased with age: 0.4%, 3% and 5.4% of subjects aged 40-59, 60-79 and 80years, respectively. One single B-cell clone was detected in 9/13 cases and two B-cell clones in 4/13 (n¼17 MBL populations). Nine MBL cell populations were CD5- (overlapping with marginal zone-derived (MZL) or lymphoplasmacytic (LPL) non-Hodgkin lymphoma -NHL- B-cells, or an unclassifiable NHL), 3 CD5-/þd and the remaining 5 were CD5þ MBL (n¼3 non-CLL-like MBL, consistent with a mantle-cell lymphoma (MCL)-like phenotype, and n¼2 CLL-like); iFISH supported the diagnosis in most cases. Twelve cases re-evaluated at 12 months showed circulating clonal B-cells, with mean levels significantly higher than those initially detected. Altogether, our results show that CLL-like MBL cases frequently show biclonality, in association with MZL-, LPL-, MCL-like, or unclassifiable phenotypic profiles. Like CLL-like MBL, the frequency of non-CLL-like MBL increases with age, with a clear predominance in males. 2

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POS-CC-10 ZAP-70 PROTEIN EXPRESSION AND ITS PROGNOSTIC VALUE IN CLL PATIENTS Jaka Lavrencak, Veronika Kloboves Prevodnik

Institute of Oncology, LJUBLJANA, Slovenia Introduction: The clinical course of chronic lymphocytic leukemia (CLL) can be indolent or very aggressive. ZAP-70 protein expression has been shown as a potentially useful prognostic marker to predict the course of the disease. The majority of studies on ZAP-70 protein expression have been performed on whole blood samples (WBS), while some on lymph nodes (LN). Aim: Firstly, to examine if ZAP-70 protein expression in fine needle aspirates (FNAs) of LN is comparable to that in WBS and secondly, to evaluate ZAP-70 index prognostic value. Materials and methods: We analysed ZAP-70 protein expression in 54 LN FNAs and 35 WBS of patients with CLL. In 21 cases, LN FNAs and WBS were from the same patient. ZAP-70 protein expression was determined by ZAP70 index, calculated on the basis of ZAP-70 protein expression in CLL cells, B and T lymphocytes. Results: ZAP-70 index was negative in 12/54 (22%) LN FNAs and 13/35 (37%) WBS, but positive in 42/54 (78%) LN FNAs and in 22/35 (63%) WBS. The correlation (rs¼0.563) of ZAP-70 indices was significant (p¼0.008) in the 21 patients with parallel LN FNAs and WBS. The clinical outcome seemed to be better in the group of patients with negative ZAP-70 index compared to those with positive ZAP-70 index. The results were the same, regardless of whether ZAP-70 index from LN FNAs or WBS was concerned. Conclusions: ZAP-70 indices were significantly higher in LN FNAs than in WBS. This is most probably associated with the ZAP-70 index calculation. Our preliminary results showed that negative ZAP-70 index could have been associated with a favourable outcome of CLL and positive ZAP-70 index with a more aggressive disease.

POS-CC-11 CYTOKINES AND LYMPHOCYTES MAY SIGNAL LIFE EVENTS IN DEPRESSION Margarida Figueiredo-Braga,1 Jose´-Enrique O’Connor,2 Fernando Mota Garcia,3 Carla Sofia Cardoso,4 Rui Mota Cardoso1 1

Medical Psychology Faculty of Medicine University of Porto, PORTO, Portugal 2 Laboratory of Cytomics, Mix Research Unit, VALENCIA, Spain 3 Clinical Pathology, Saõ Marcos General Hospital, Braga, BRAGA, Portugal 4 School of Criminology, Faculty of Law, University of Porto, PORTO, Portugal A crucial role has been attributed to stressful Life Events in depressive disorders and their quantitative and qualitative characterization proved to help understand psy-

chiatric disease. Cytokine dysfunction has been related to depression. In a previous study1 aimed at characterizing immune system involvement in depression, cytokines were determined in plasma by a multiplex system technology in 100 females. Life Events number was measured to relatively short (1 month) or long time (6 months) occurrence, impact and quality. IFN-, IL-1, IL-2, IL-6, IL-4 and IL-5 correlated negatively with only the number of long-term Life Events in depressed patients. A positive association was seen, however, between the impact of those Life Events IFN-, IL-2 and IL-6 levels. Statistically significant negative correlations were also detected between lymphocyte population numbers (total lymphocyte, CD3, CD4, CD8, and CD19) and the impact of short-term Life Events. The results highlight the possibility of ‘‘specific’’immunological responses to psychological challenges. Persistent challenges could trigger enhanced cytokine expression associated with higher impact of the event. Lymphocyte counts and cytokine determination may therefore represent disrupted immune responses in depression. Reciprocally, we have shown before1 that anxiety seems to relate to immunological markers. Both observations stress the potential importance of defining the reciprocal interactions between the immune system and the expression of depression., 1Figueiredo-Braga et al. 2009 Cytokines and Anxiety in Systemic Lupus Erythematosus (SLE) Patients Not Receiving Antidepressant Medication. Ann N Y Acad Sci 1173;286-91.

POS-CC-12 ANTIFUNGAL SUSCEPTIBILITY OF CANDIDA PARAPSILOSIS AND CANDIDA ALBICANS BLOODSTREAM ISOLATES TO AMPHOTERICIN B DETERMINED BY FLOW CYTOMETRY METHODOLOGY Lourdes Cordoń,1 Eva M. Gonza´lez,2 Emilia Cantoń,2 Amparo Sempere,1 Isidro Jarque,1 Javier Pemań2 1

Department of Hematology and Hemotherapy. Hospital Universitario La Fe, VALENCIA, Spain 2 Department of Experimental Microbiology. Hospital Universitario La Fe, VALENCIA, Spain Introduction: Opportunistic fungal infections are a leading cause of morbi-mortality in immunosuppressed hosts. The standardized techniques for antifungal susceptibility testing (AST) require between 24-48 h. Therefore, it is of great importance to develop a faster method. Flow cytometry (FC) has been applied for AST and could be an alternative. Objectives: To develop a method for determining the minimum inhibitory concentration (MIC) of amphotericin B (AMB) by FC using FUN-1 (metabolic activity marker) and to compare the results with the MICs obtained by CLSI dilution reference method (DM). Materials and methods: A total of 10 Candida parapsilosis and 9 C. albicans strains from blood cultures were tested. C. parapsilosis ATCC 22019 was used as quality control. DM was performed according to document M27-A3. The FC method was based on a 4-h incubation of yeasts with twofold dilutions of AMB. Metabolic activity was meas-



ured every 30 min with FUN-1. FC MIC was the lowest concentration that produced a decrease in metabolic activity 90%. Results: Metabolic activity was time and concentration dependent and 3 h was enough to read FC MIC. FC MICs for C. albicans were 0.125 mg/L vs. 0.125-0.5 mg/L by DM. For C. parapsilosis ranges were 0.125-0.25 mg/L vs. 0.125-0.5 mg/L. We did not observe any categorization discrepancy between methods (all isolates were inhibited by 0.5 mg/L of AMB). Conclusion: As FC is an appropriate method for testing susceptibility of yeasts to AMB, this shorter time-consuming method (3h) could be employed as a substitute for DM. In addition, FC could give single day result availability. Further studies to assess the correlation between methods are warranted.

POS-CC-13 FLOW CYTOMETRY PROTOCOL OF NK AND NKT CELLS VALIDATION Evgenia Konsta,1 Katherina Psarra,2 Violetta Kapsimali,3 Michael Koupparis,4 Chryssa Papasteriades2 1

Second Department of Internal Medicine, Division of Hematology, ATTIKON Hospital, ATHENS, Greece 2 Department of Immunology and Histocompatibility, Evangelismos Hospital, ATHENS, Greece 3 Laboratory of Microbiology,Medicine School, National and Kapodistrian University, ATHENS, Greece 4 Department of Chemistry, National and Kapodistrian University of Athens, ATHENS, Greece Introduction: Flow Cytometry has become an integral part of clinical pathology. The need to have instrument and methodology quality control procedures becomes paramount. Materials and method: In order to validate protocol CD3-ECD/CD56-PC5/CD2-FITC/CD16-PE of NK and NKT cells, Immuno-TrolTM cells, a peripheral blood sample from a healthy individual and one patient with NK lymphocytosis were chosen. Repeatability (n¼6), reproducibility (n¼4), detection limits, accuracy and robustness of the protocol were defined. Results: Concerning repeatability, percentages of lymphocytes (CV¼0.66-2.7%), NK cells (CV¼3.5-15%) and their subsets [CD3-CD56þ(CV¼4.9-11%), CD3-CD16þ(CV¼1.88.2%)] were preserved while differences were observed in NKT cells (CV¼3.1-33%) and their subsets [CD3þCD56þ(CV¼2.6-24%), CD3þCD16þ(CV¼8.6-36%)]. Concerning reproducibility, NK, NKT cells and their subsets percentages were acceptable until the third day. ImmunoprepTM was found suitable for whole blood lysis, whereas 75% of the indicated quantity of Monoclonal Antibodies (MoAbs) and 5 min incubation time of the sample with MoAbs have not affected the results, consisting thus the optimum conditions of sample preparation. Dilution 1:16 of control sample was found appropriate to define protocol detection and quantification limits. Method accuracy was checked by comparing measurements of the same sample in two different flow cytometers.


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Conclusion: Protocol validation of NK and NKT cells provided important information concerning both clinical and investigation purposes. Continuous improvement in the quality should be a driving force in every laboratory.

POS-CC-14 IMMUNOPHENOTYPING OF MATURE B-CELL NEOPLASMS CORRELATION BETWEEN FLOW CYTOMETRY AND IMMUNOHISTOCHEMISTRY Nada Kraguljac Kurtovic, Tijana Dragovic-Ivancevic, Vesna Knezevic, Maja Perunicic Jovanovic, Mirjana Gotic, Biljana Mihaljevic

Clinical Center of Serbia / Institute of Hematology, BELGRADE, Serbia Background: Immunophenotyping by flow cytometry (IFC) and immunohistochemistry (IHC) define the appropriate immunophenotype of malignant B-cells and predict the type of mature B-cell neoplasm. These two methods are usually used as complementary tools in diagnostic purposes. Aims: Correlation of immunophenotypic results regarding expression of three antigens, CD20, CD23, CD5, simultaneously explored by IFC and IHC. Methods: During 2009, wWe analyzed 27/152 adult patients (pts) with diagnosis de novo mature B-cell neoplasm, immunophenotyped in parallel by IFC and IHC. IFC was performed by using standard 3- and 4-color staining protocols on whole peripheral blood specimens. IHC was performed on paraffin-embedded bone marrow biopsy specimen by using standard methodology. Results: CD20 was positive in all (27/27) tested pts by both methods (100% concordance), and was characterized by CD20 high expression pattern in all pts, as detected by IFC. CD23 was positive in 9/18 pts, only by IFC, with CD23low expression pattern in 6/9 pts and CD23medium pattern in 3/ 9 pts, whereas 1/18 pts was positive only by IHC. CD23 was negative in 8/18 pts by both methods (44% concordance). CD5 was positive in 7/16 pts, only by IFC, with CD5low expression pattern in 3/7 pts and CD5 medium/high patterns in 4/7 pts. The rest 9/16 pts were either CD5 positive or CD5 negative by both methods (56% concordance). Conclusion: The essential advantage of IFC over IHC lies in its higher sensitivity to detect antigens with lower expression levels (CD23 and CD5). Differences in sensitivity between IFC and IHC may have influence on establishing the final diagnosis of mature B-cell neoplasm.

POS-CC-15 ESTABLISHING OF STEM CELL CHARACTERIZATION ASSAYS BY FLOW CYTOMETRY. Somchai Sangkitporn, Siripakorn Sangkitporn, Kanyarat Rattanakittisophon, Apichat Chotchusri, Chonlada Yodtup, Sawitree Duangruang, Acharaporn Dumbua, Suppaluk Buasrikaew, Patcharaporn Boonchu

Clinical Research Center, NONTHABURI, Thailand Stem cell-based therapies are being investigated to develop new methods to repair or replace tissues or damaged

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cells through injuries or diseases. With increasing stringent regulatory requirement, quality control is playing an important role in stem cell clinical studies. Several cellular markers indicative of either cell type, pluripotency or lineage commitment can be used to establish characterization assay for monitoring stem cell manufacturing process and product release testing. This study was performed to establish the characterization assays of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) by flow cytometry. According to their different surface markers; HSC, MSC and EPC were characterized as CD34þ/ CD45-, CD105þ/ CD34- and CD34þ/ CD133þ/ VEGFR2þ, respectively. Validation parameters, including accuracy and precision were used to assess the analytical performance. Variabilities of these assays were quite low. The narrow scatter of the results confirms the reproducibility of the assay. Due to the lack of specific reference materials, the inter-laboratory comparisons were done to estimate accuracy. All results were correlated well with those of the reference laboratory. These characterization assays were used to identify HSC, MSC and EPC in bone marrow mononuclear cell preparation in the clinical study of post-infarction heart failure in children. In conclusion, the establishing of stem cell characterization assay is one of the most important steps to realizing that stem cell which could provide the basis of clinical treatment are qualified for use under properly controlled conditions.

POS-CC-16 HITALERT ASSAY, A NOVEL FUNCTIONAL FLOW CYTOMETRIC ASSAY TO DETERMINE THE PRESENCE OF HIT ANTIBODIES Henk SP Garritsen,1 S Flemisch,2 E. De Boef,3 J.H.N. Schuitemaker,3 A. Konitzke,2 N. Legath,2 W. Eberl4 1

Inst. for Clinical Transfusion Medicine, Sta¨dtisches Klinikum Braunschweig gGmbH, BRAUNSCHWEIG, Germany 2 Instituer for Clinical Transfusion Medicine,Sta¨dtisches Klinikum Braunschweig, BRAUNSCHWEIG, Germany 3 IQ Products, GRONINGEN, The Netherlands 4 Dept. of Pediatrics, Sta¨dtisches Klinikum Braunschweig gGmbH, BRAUNSCHWEIG, Germany Introduction: HIT is an immune-mediated complication of heparin treatment. It is advisable to combine a heparin/PF4- antibody test with a functional assay and clinical evaluation. We evaluated a novel flow cytometric functional test: HITAlert. The HITAlert platelet activation assay detects antibodies that recognize heparin complexes independent of a second molecule and it only shows those antibodies capable of inducing activation of the platelets. Methods: We analysed 151 consecutive patients referred for HIT testing in our laboratory. HIT evaluation consisted of a PF4 IgG-ELISA test (GTI) the HITAlert test (IQ Products) and a clinical score (m4T’s). Results: Of the 151 clinical samples submitted for HIT, 117 were negative in the PF4 IgG-ELISA test (GTI), 34 were positive. Of these 34 Elisa positive samples, 21 samples were weakly positive and 13 samples were clearly positive.

In the HITAlert assay, 136 samples were negative and 15 samples were positive. 14 of these 15 samples were included in the group of 34 ELISA positive samples. 9 HITAlert positive samples were in the group of 13 ELISA clearly positive samples. With regard to the m4T-score, the 9 samples which were clearly ELISA positive and HITAlert positive had a high clinical score. The 4 ELISA weakly positive, HITAlert positive samples all had a low/moderate clinical score. Conclusions: In our opinion, HIT diagnosis consists of a combination of a clinical score, a PF4 IgG antibody screening test and a sensitive functional test which is nonradioactive and readily available, such as the one evaluated here.

POS-CC-17 SUPPRESSION OF MITOGEN-INDUCED PROLIFERATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS BY H.PYLORI IN THE PRESENCE OR ABSENCE OF LACTIC ACID BACTERIA Izabela Kubiszewska,1 Anna Helmin-Basa,1 Lidia Gackowska,1 Milena Urbanska,1 Andrzej Eljaszewicz,1 Malgorzata Wiese,1 Ilona Motyl,2 Katarzyna Slizewska,2 Adriana Nowak,2 Zdzislawa Libudzisz,2 Jacek Michalkiewicz1 1

Department of Immunology, Collegium Medicum, Nicolaus Copernicus University, BYDGOSZCZ, Poland 2 Institute of Technology Fermentation and Microbiology, Technical University, LODZ, Poland The aim: Data indicate that Helicobacter pylori (H.p.) may have not only up but also down regulatory effects on immune responses. The aim of this study was: 1) to examine suppressive mechanism of H.p. on mitogen-induced cell proliferation and 2) to estimate immunomodulatory effect of lactic acid bacteria (LAB) on pathogen-induced cell proliferation. Materials and methods: PBMCs were induced (72h) by Gramþ strains (L.plantarum 0864, L.plantarum 0862), H.p. and LAB/H.p. in the presence of anti-CD3 Ab (3H thymidine incorporation). Levels of IL-12p40, IFN- and IL-10 in supernatants were measured by ELISA. CD4þ and CD8þ proliferation (CFSE), apoptosis (Annexin-V/7-AAD) and Treg cells (CD4þ/CD25þ/CD127-) were measured using FACScan. Results: H.p., as well as LAB strains, significantly decreased proliferation of PBMCs. LAB were equipotent in suppressing PBMC, while H.p. was less effective. Suppression applies to both the CD4þ and CD8þ cells population. There were no differences in apoptosis induced by a/CD3 and bacteria strains. Only L.plantarum 0864 (single strain and with H.p.) increased apoptosis. LAB induced secretion of large amounts of IL-12p40, while H.p. induced much less IL-12p40. High secretion of IL-12p40 was also observed after stimulation with mixture LAB/H.p. (vs. H.p. and a/CD3). Conclusions: Antiproliferative capacity of H.p. seems to be not only selectively related to pathogen but also to other bacteria, such as LAB strains. Our data showed that this effect is not correlated to cell apoptosis as well as to



induction of Treg cells. Results suggest that the major cause of this suppressive effect may be dependent on monocytes rather than T cells. The study was supported by grant from Ministry of Science and Higher Education 1321/P01/2006/30.

POS-CC-18 FLOW CYTOMETRY (FCM) DETECTION OF EPITHELIAL CELLS IN BRONCHOALVEOLAR LAVAGE Julia Illan,1 Cristina Serrano,2 M. Ańgeles Escudero,1 Susana Castanõń,2 A. Isabel Gutie´rrez,3 Antonio Contreras,3 Eva Romo,1 Dolores Subira´4 1

Unilabs Diagno´sticos S.L., MADRID, Spain Fundacioń Jimeńez Dıáz, MADRID, Spain 3 Hospital de Torrevieja, TORREVIEJA, Spain 4 H.U. Guadalajara, GUADALAJARA, Spain The distribution of hemopoietic populations in bronchoalveolar lavage (BAL) has well-known prognostic or diagnostic value in patients with interstitial lung diseases, sarcoidosis and opportunistic infections. However, description of epithelial cells in BAL might be related to contamination with bronchial secretions or lung neoplasia. Aim: To establish a normal range of epithelial cells in BAL not associated with epithelial neoplasia. Methods: 30 BAL samples from 29 patients (median age: 70, range 23-87), diagnosed with non-tumoral lung diseases. Samples were processed within 24 hours after extraction and studied by FCM using a 4-color panel of direct fluorescent monoclonal antibodies. A Mab against the antigen EpCAM was used to identify epithelial cells. CD3, CD15, CD33, CD45, and HLA-DR were used to identify lymphocytes, eosinophils, neutrophils and macrophages. Results: Median percentage of EpCAMþ cells was 0.32%, (range 1% of EpCAMþ cells. Median percentages (range) of the remaining cells were: 29.1% (0.4-77%) neutrophils; 0.3% (0-1.4%) eosinophils; 58.4% (3-95%) macrophages; 10.2% (2-40%) lymphocytes. The cytologic examination of these BAL confirmed the lack of epithelial malignant cells. Conclusion: According to our small series, >1% of epithelial cells were detected in BAL from patients with a wide range of lung diseases. Furthermore, description of 4% of epithelial cells was not associated with epithelial malignancies. No correlation was found between the rate of EpCAMþ cells, hemopoietic BAL cells or lung disease. This study should be expanded to describe the rate of EpCAMþ cells in patients suffering from epithelial lung cancers. 2

POS-CC-19 ACUTE RETROVIRAL SYNDROME (ARS) WITH UNUSUAL INCREASE IN ‘LARGE GRANULAR’ LYMPHOCYTES Lais Pinto de Almeida,1 Annelise C. Wengerkievicz,1 Fla´via D. Xavier,2 Fernanda M.M. Ramalho,3 Kleiner V. Pinheiro,1 M. Mirtes Sales1 1

Hospital das Clıńicas da Faculdade de Medicina da USP, SAÕ PAULO, Brazil


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2

Instituto do Caˆncer do Estado de Saõ Paulo - ICESP, SAÕ PAULO, Brazil 3 Hospital Santa Marcelina, SAÕ PAULO, Brazil Introduction: The ARS period, stretching between the infection with the Human Immunodeficiency Virus (HIV) through complete seroconversion, typically presents in 3050 days as a mononucleosis-like syndrome. Up to 90% of patients show any hematological abnormality. Case report: A 46-year-old male came to hospital with fever, myalgia, headache, cough, diarrhea and weight loss for over a month. He was febrile, with a painless splenomegaly and adenopathy. General tests and serologies were carried out. Inflammatory markers were above the normal range. The blood count showed lymphocytosis (23,900/ mm3) with 32% of ‘‘large granular’’ morphology. HIV-ELISA and Western Blot were positive, and the viral load was 8,352 copies/mL. Infections by other viruses or opportunistic agents were ruled out. Because of lymphocytosis, the immunophenotypic study of peripheral blood was performed for lymphoproliferative disease (LPD), which showed increased T lymphocytes, CD3 ¼ 96% (22176/ mm3), with predominance of CD8 subpopulation (93%) and inversion of CD4/CD8 ratio (0.03). B and NK lymphocytes were significantly decreased. In order to discard CD8-positive LPD, TCR-clonality by molecular technique was performed but was negative. The patient is currently in follow up without meeting the criteria for antiretroviral therapy. Conclusion: In ARS there is an inversion in CD4/CD8 ratio and activation of the entire Immune System. The patient described showed marked lymphocytosis that evoked the investigation of LPD, which is prevalent in HIV infection. The clinical outcome correlated with laboratory tests and therefore led us to conclude the diagnosis of ARS.

POS-CC-20 FLOW CYTOMETRIC DETECTION OF BCR-ABL HYBRID PROTEINS IN CML PATIENTS IN COMPARISON TO RT-PCR-BASED APPROACHES Margarita Guenova, Gueorgui Balatzenko, Nikolay Stoyanov, Vassil Hrischev, Branimir Spassov, Penka Ganeva, Georgi Michailov

National Haematological Hospital, SOFIA, Bulgaria Background: The detection of BCR-ABL fusion gene is important for the diagnosis of chronic myeloid leukemia (CML) and for treatment effectiveness evaluation. Routinely, cytogenetics, FISH or PCR are used to detect the aberration at chromosome, DNA or RNA level. Recently, immunological flow cytometry (FCM) approach has been developed at the protein level. Aim: We applied a novel FCM assay for the detection of BCR-ABL hybrid protein in comparison to PCR methods. Materials and methods: Cell lysates from BCRABL(þ) CML pts were tested. K562 and HL60 cell lines were included as a (þ) and (-) control. A bead-bound antiBCR catching antibody and a fluorochrome-conjugated antiABL detection antibody were applied to detect the hybrid protein by FCM (BD). All samples were also studied by con-

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ventional and quantitative Q-RT-PCR in order to identify the fusion transcripts presence, type and level. Results: All pts samples and cell lines with molecular evidence of BCR-ABL showed (þ) fluorescent signal by FCM. Dilution experiments revealed sensitivities of 1-0,5%. Using conventional RT-PCR, all clinical samples at diagnosis showed strong reaction (not allowing for the distinction of cases with various levels of expression), while Q-RT-PCR showed variations in the BCR-ABL/ABL ratio from 0,65-0,95. FCM data also differed from low positive to high positive samples (K562, small proportion of chronic phase CML patients). Conclusion: FCM showed concordant results with the routine molecular approaches. Yet the sensitivity is insufficient for monitoring minimal residual disease and further studies are warranted. Acknowledgements to the National Research Fund (BIn03/06; VUL315/07, CVP01/0119).

POS-CC-21 LYMPHOCYTE POPULATIONS AND SERUM CYTOKINES RECOVERY SIX MONTHS AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELLS TRANSPLANTATION Margarita Guenova,1 Antoaneta Michova,2 Maria Nikolova,2 Lidia Garcheva,1 Penka Ganeva,1 Georgi Michailov,1 Hristo Taskov,2 Margarita Guenova1 1

National Haematological Hospital, SOFIA, Bulgaria National Center of Infectious and Parasitic Diseases, SOFIA, Bulgaria Background: Immune recovery after autologous hematopoietic stem cell transplantation (aHSCT) is characterized by specific reconstitution kinetics of leukocyte populations and their functions. Aim: To study the recovery dynamics of lymphocyte populations 2 years after aHSCT, in parallel with serum cytokine levels. Materials and methods: Peripheral blood and serum of 24 patients undergoing aHSCT were studied before transplantation (M0), and after 2, 6, 9, 12, 18 and 24 month (M2 - M24). Lymphocyte populations were studied by flow cytometry - FCM (BD FACSCantoII, BD). Serum cytokines levels were determined by cytokine bead array (human Th1/Th2 kit; BD). Results: The lymphocyte rapidly increased at M2 and showed a slight decrease at M24. T cells recovery showed a sharp increase at M2, decrease at M12 and enhancement to 74% (775 cell/l) at M24. The NK cells gradually increased after M1, reaching 14% (130 cells/l) at M24. A very low level of B cells was detected after aHSCT rising at M12, followed by a decrease to 9% (167 cell/l) at M24. The levels of IL-2, IL-4, IL-5 varied insignificantly and remained just above the detection limit. At M24 the mean IFN- concentration was 16.65 pg/ml, IL-10 - 6.21 pg/ml after the increase at M9, and TNF- - 4.03 pg/ml. TNF- significantly correlated with IFN- and IL-10, and with a proportion of B cells while a negative correlation was detected with naı¨ve CD4þ and CD8þ cells and central-memory CD4þ cells. 2

Conclusion: Post aHSCT immune recovery is a long and multifactorial process. FCM reliably demonstrates the dynamics of the reconstitution and promotes for immunological monitoring of transplanted patients. Acknowledgements to National Research Fund.

POS-CC-22* B CELL SUBSETS IN CEREBROSPINAL FLUID OF PATIENTS WITH HU-ANTIBODY ASSOCIATED PARANEOPLASTIC NEUROLOGICAL SYNDROMES: PREDOMINANCE OF MEMORY B CELLS. Adriaan H.C. de Jongste, Jaco Kraan, Patricia D.M. van den Broek, Peter A.E. Sillevis Smitt, Jan W. Gratama

ErasmusMC/Daniel den Hoed, ROTTERDAM, The Netherlands In the anti-Hu antibody (Hu-Ab) associated paraneoplastic neurological syndrome (Hu-PNS), high titers of Hu-Ab are found in peripheral blood. Intrathecal antibody production has been shown and B cells have been observed in cerebrospinal fluid (CSF), brain and nerve tissue. A causal role of HuAb in Hu-PNS is unlikely as Hu-Abs did not cause neurologic symptoms in animal experiments. However, B cells might play a role in the pathogenesis of Hu-PNS by antibody independent mechanisms. We examined the phenotype of different B cell subsets in CSF and blood of Hu-PNS patients with progressive symptoms (n¼9) and age-matched controls with a negative neurological anamnesis (n¼43) by 6-color flow cytometry. The peripheral blood of Hu-PNS patients contained predominantly naı¨ve B cells (CD19þCD27-CD38-/ þCD138-), similar to controls. Five out of 9 CSF samples of Hu-PNS patients contained sufficient B cells for further differentiation, compared to 0 out of 43 control samples. In HuPNS CSF, most B cells were CD19þCD27þCD38-/þCD138memory cells (54%, 40-82%; all data: median, range). Of these cells, median 52% (range 9-59%) were class switched memory cells (CD19þCD27þCD38-/þCD138-sIgD-sIgM-). Naı¨ve B cells (CD19þCD27-CD38-/þCD138-) made up 18%, 0-42% and plasmablasts (CD19þCD27þCD38þþ) made up 15%, 14-27% of the total CD19þ B cell population. Most plasmablasts expressed CD138 (80%, 75-97%). Plasma cells without CD19 expression (CD19-CD38þþCD138þ) were almost absent. We conclude that in CSF of Hu-PNS patients most B cells are memory cells. Although these B cells could have resulted from earlier infections, their absence in controls suggest that they are involved in the pathogenesis of the Hu-PNS.

POS-CC-23 APOPTOSIS OF DIFFERENT T LYMPHOCYTE AND MONOCYTE SUBSETS IN THE PERIPHERAL BLOOD OF CHILDREN WITH H. PYLORI INFECTION. Anna Helmin-Basa,1 Izabela Kubiszewska,2 Lidia Gackowska,2 Andrzej Eljaszewicz,2 Grazyna Mierzwa,3 Anna Szaflarska-Poplawska,3 Mieczyslawa Czerwionka-Szaflarska,3 Andrzej Marszalek,4 Jacek Michalkiewicz2 1

Department of Immunology, Collegium Medicum Nicolaus Copernicus University, BYDGOSZCZ, Poland


ESCCA AND SIC 2010 ABSTRACTS 2

Dept. Immunology, CM N. Copernicus University, BYDGOSZCZ, Poland 3 Dept. Pediatrics, Allergology and Gastroenterology, CM N. Copernicus University, BYDGOSZCZ, Poland 4 Dept. Clinical Pathomorphology, CM N. Copernicus University, BYDGOSCZ, Poland H. pylori infection induces apoptosis of the epithelium and lymphocytes in gastric mucosa. The impact of H. pylori infection on apoptosis of different cell subsets has not been studied in children. Objective: The aim of this study was to evaluate the apoptosis of different monocyte and T lymphocyte subsets in the peripheral blood of children with H. pylori infection, children with gastritis where H. pylori infection was excluded, as well as control children. Additionally, circulating cell apoptosis was correlated with gastric inflammation scores. Materials and methods: The dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify cell apoptosis in combination with the surface antigen expression (CD3, CD4, CD8, CD45RA, CD45RO, HLA-DR, CD16). Results: Increased apoptosis of T helper and cytotoxic lymphocytes were observed in H. pylori-infected children in comparison with non-infected children with gastritis and in control children. Additionally, elevated apoptosis of naive and memory T cells as well as pro-inflammatory monocytes bearing HLA-DRþ/CD16þ phenotype were found in children with gastritis, regardless of H. pylori infection. No correlation between apoptosis of circulating cells and gastric inflammation scores was noted. Conclusions: H. pylori infection is associated with high apoptosis of T helper and cytotoxic lymphocytes. However, gastritis induces apoptosis of pro-inflammatory monocytes, as well as of naive and memory T cells, regardless of H. pylori infection. This work was supported by grant UMK 20/2010

POS-CC-24 VALIDATION OF A NEW FLOW CYTOMETRY-BASED METHOD TO DISCRIMINATE MALIGNANT FROM NORMAL STEM CELLS IN CML. Regina Garcia, Santiago Del Castillo, Laura Entrena, Ana Isabel Rossell, Arturo Campos, Maria Paz Queipo de LLano, Gemma Ramirez

Hospital Virgen de la Victoria, MALAGA, Spain Introduction: Previously, leukemic stem cells in Chronic Myelogenous Leukemia could only be identified indirectly by using culture techniques. Here, we have used a new flow cytometric approach that enables CML stem cells to be directly distinguished from their normal counterparts within single patient samples. The fact that CD34þCD38- stem cells could be discriminated from normal stem cells by high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. Materials: A total of 5 patients and health controls were included in this study. 3 cases at diagnosis and 3


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months after treatment with Imatinib. 1 case after 3 and 6 months with Imatinib and in the last one, in a resistant one. Results: Flow cytometry data confirmed the different expression of CD90 in patients when the levels of BCR-ABL are lower. Conclusions: Our results confirm the data of Jeroen and this new technique will expand our possibilities of identifying new CML stem cell specific targets and may further improve efficacy assessment of CML treatment.

POS-CC-25 EXPRESSION OF ADHESION AND ACTIVATION MOLECULES ON CIRCULATING MONOCYTES IN CHILDREN WITH H. PYLORI INFECTION. Anna Helmin-Basa,1 Grazyna Mierzwa,2 Jacek Michalkiewicz,3 Lidia Gackowska,3 Izabela Kubiszewska,3 Andrzej Eljaszewicz,3 Grazyna Bala,2 Anna Szaflarska-Poplawska,2 Andrzej Marszalek,4 Mieczyslawa Czerwionka-Szaflarska2 1

Department of Immunology, Collegium Medicum Nicolaus Copernicus University, BYDGOSZCZ, Poland 2 Dept. Pediatrics, Allergology and Gastroenterology, CM N. Copernicus University, BYDGOSZCZ, Poland 3 Dept. Immunology, CM N. Copernicus University, BYDGOSZCZ, Poland 4 Dept. Clinical Pathomorphology, CM N. Copernicus University, BYDGOSCZ, Poland Objective: The aim of study was to assess the cell surface expression of adhesion (CD11a, CD11b, CD11c, CD18, CD54, CD58) and activation (CD14, HLA-DR, CD16) molecules on the circulating monocytes in children affected by H. pylori infection and gastritis. Additionally, monocyte surface receptor expression was correlated with gastric inflammation scores. Materials and methods: Forty-seven children with H. pylori infection (25 children after eradication therapy), 26 children with gastritis where H. pylori infection was excluded, as well as 21 control children matched for gender and age were studied. H. pylori status was assessed by 3 different methods (C13 urea breath test, rapid urease test and histology). Monocyte surface receptor expression were analyzed by flow cytometry. Results: H. pylori-infected children and children after failure eradication therapy differ significantly in the expression of adhesion and activation molecule on circulating monocytes. There were decreased both the proportion of CD11c- and CD14-bearing monocytes and fluorescence intensity for CD11c and CD14 molecules in children after failure eradication therapy. Low percentage of HLA-DR positive monocytes and high population of CD16 positive monocytes were also observed. Additionally, expression of CD11b and CD18 leucocytic integrins on monocytes were decreased. Compared to the control group, low percentage of CD11a positive monocytes were noted in children regardless of H. pylori infection and eradication therapy. Conclusion: H. pylori eradication therapy significantly decreased expression of CD11b, CD11c and CD18 beta2integrins on circulating monocytes in children.

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POS-CC-26 OXIDATIVE STRESS IN CML STEM AND PROGENITOR CELLS CORRELATES WITH RESPONSE TO IMATINIB Regina Garcia, Santiago Del Castillo, Laura Entrena, Ana Isabel Rossell, Arturo Campos, Maria Paz Queipo de LLano, Gemma Ramirez

Hospital Virgen de la Victoria, MALAGA, Spain The BCR/ABL kinase alters the oxidative environment in chronic myelogenous leukemia (CML) cells, but the consequences of the increased reactive oxygen species (ROS) levels on signaling pathways remain unknown. Increased intracellular peroxides in progenitor cells have been linked to DNA damage, and promote self-mutation which subsequently renders the leukemia cells resistant to Imatinib, leading to failure to eliminate them. Material and Methods: A total of 5 patients and health controls were included in this study: 3 cases at diagnosis and 3 months after treatment with Imatinib; 1 case after 3 and 6 months with Imatinib and the last case was a resistant one. Oxidative stress biomarkers were studied by flow cytometry in the bone marrow progenitor cells. Results: Not only levels of BCR-ABR and the number of CML stem cells were lower with treatment, but also the oxidative stress biomarkers were lower in normal and CML stem cells in patients with response to Imatinib. However, in the resistant one, the ROS levels were higher in all the stem cells Conclusion: The levels of oxidative stress are significantly higher in CML cells than in healthy subjects and correlate with tumor burden as well as the response to Imatinib. This data demonstrate a role of oxidative stress in restoring normal hematopoiesis in CML patients and probably in Imatinib resistance

POS-CC-27 EXPRESSION OF COAGULATION FACTOR XIII SUBUNIT A IN ACUTE PROMYELOCYTIC LEUKEMIA Agnes Simon,1 Zsuzsa Bagoly,2 Zsuzsanna Hevessy,1 Eva Katona,2 Gyo¨rgy Vereb,3 Laszlo Szerafin,4 Laszlo Muszbek,2 Janos Kappelmayer1 1

Dept. of Clinical Biochemistry and Molecular Pathology, University of Debrecen, DEBRECEN, Hungary 2 Clinical Research Center, Thrombosis and Haemostasis Research Group of the HAS, DEBRECEN, Hungary 3 Dept. of Biophysics and Cell Biology, DEBRECEN, Hungary 4 Dept. of Hematology, Josa Andras Teaching Hospital, NYIREGYHAZA, Hungary Background: Leukemic cells often express markers which are not characteristic of their particular cell lineage. In this study we identified the ‘‘A’’subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/ macrophages. Furthermore, more recently, we described the presence of FXIII-A in leukemic lymphoblasts.

Materials and Methods: Here, we studied 13 patients with a follow-up of up to 108 months and investigated their bone marrow and peripheral blood samples by 3-color flow cytometry upon diagnosis. We detected FXIII-A also by ELISA, Western-blot and confocal laser scanning microscopy. Results: In most samples, using flow cytometry, FXIII-A was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD13þ/CD33þ/CD117þ/cyMPOþ and HLA-DR-/CD34-/CD14-/CD15-). Platelet markers GPIIb and GPIX were negative, and FXIII-A was detected in the cytoplasm of the cells by confocal microscopy. FXIII-A content was quite high as measured by ELISA. By Western blot analysis, we could identify FXIII-A in the native 82 kD form and in cleaved forms, corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. We also found that the 9 FXIII-A positive APL patients all responded to treatment and are alive, while the 4 FXIII negative cases have all died. Conclusions: This novel expression site of FXIII-A can be considered as a leukemia-associated immunophenotype in AML M3.

POS-CC-28 FLOW CYTOMETRY CD45-NEGATIVE B-NHL: A CASE REPORT OF A DIFFUSE LARGE B-CELL LYMPHOMA. Carlos Palmeira, Maria Emı´lia Sousa, Ineˆs Godinho, Ana Marta Pires, Carlos Mendes, Gabriela Martins

Cytometry Laboratory, IPO, PORTO, Portugal B-cell non-Hodgkin lymphomas (B-NHL) typically express CD45, a transmembrane protein tyrosine phosphatase, also known as the leukocyte-common antigen. Absent CD45 expression, evaluated by flow cytometry, is a very unusual phenotype in B-NHL. Here we report a CD45-negative case of a diffuse large Bcell lymphoma (DLBCL) in a 65 year old male patient. Immunophenotyping and DNA content analysis were performed by multiparametric flow cytometry in lymph node specimens. Malignant B-lymphocytes were CD5-, CD10þ/þþ, CD11c-, CD19þ, CD20þ/þþ, CD23-, CD45-, CD79bþþ/ þþþ, BCL2 without overexpression, FMC7þþ, IgMþþ/ þþþ, and Lambda light chain restriction. This pathological cellular population showed near-diploid DNA content, with a high proliferative rate. CD45-negative phenotype has been described in rare cases of large B-cell lymphoma (LBCL) subtypes with extranodal involvement, anaplastic lymphoma kinase positive LBCL and a DLBCL. However, this case only showed lymph node and blood marrow involvement.

POS-CC-29 MULTICOLOR FLOW CYTOMETRY FOR DISTINCTION OF NORMAL B-LINEAGE CELLS AND ACUTE LYMPHOBLASTIC LEUKEMIA BLASTS. Lukasz Sedek,1 Joanna Bulsa,1 Alicja Sonsala,1 Iwona Malinowska,2 Igor Olejnik,3 Maciej Niedzwiecki,4 Benigna Konatkowska,5 Grazyna Sobol,6 Joanna Trelinska,7 Katarzyna Muszynska-Roslan,8


ESCCA AND SIC 2010 ABSTRACTS 2

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Michal Matysiak, Maria Wieczorek, Wojciech Mlynarski, Bogdan Mazur,1 Tomasz Szczepanski1 1

Department of Pediatric Hematology and Oncology, Medical University of Silesia, ZABRZE, Poland 2 Department of Pediatric Hematology and Medical Oncology, Medical University in Warsaw, Poland 3 Department of Hematology and Oncology, Pediatric Hematology and Oncology Center in Chorzow, Poland 4 Department of Pediatric Hematology, Oncology and Endocrinology, Medical University in Gdansk, Poland 5 Department of Hematology, Oncology and Pediatric Transplantology, Medical University in Poznan, Poland 6 Department of Oncology, Hematology and Chemotherapy of Clinic of Pediatrics, Medical University of Silesia, KATOWICE, Poland 7 Department of Pediatric, Oncology, Hematology and Diabetology, Medical University in Lodz, Poland 8 Department of Pediatric Oncology, Medical University in Bialystok, Poland Multi-color flow cytometry is a basic diagnostic investigation in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). It allows establishment of the exact phenotype of malignant cells in bone marrow sample in a quick and reproducible manner. The aim of the study was to compare the detailed phenotype of normal B-cell precursors (3 types of hematogones), mature B-cells and BCP-ALL blasts. The assessed antigens were: CD10, CD45, CD34, TdT, CD20, CD22 and CD38. The study group consisted of 154 children with CD10þ BCPALL, 15 with CD10- BCP-ALL and 31 children with non-malignant minor hematological disorders (age range 0-18 years). The expression of antigens on bone marrow cells was assessed with 12-score scale based on fluorescence intensity and homogeneity in cases where the fluorescence was spread over more than one level of intensity. Additionally the mode of non-homogeneous expression (continuous or separated) was distinguished, generating 21 possible states of expression of single antigen on examined cells. The results showed moderate heterogeneity among the 3 types of hematogones and mature lymphocytes B. The mean coefficient of phenotype concordance for all 4 types of normal cells reached 55.7%. In contrast to BCP-ALL CD10þ and CD10- patients respectively, 151/154 and 15/15 different phenotypes were obtained (coefficient of phenotype concordance 1.9% and 0%, respectively). The applied approach enabled a clear distinction between the phenotype of blasts in BCP-ALL and their normal counterparts. Assessment of expression states of each individual antigen revealed significant differences among particular types of normal cells as well as between normal cells and leukemic blasts.

POS-CC-30 ACUTE LEUKEMIA OF AMBIGUOUS LINEAGE: A CASE REPORT OF MIXED T/MYELOID PHENOTYPE. Gabriela Martins,1 Ineˆs Godinho,1 Maria Emı´lia Sousa,1 Carlos Palmeira,1 Ana Marta Pires,1 Carlos Mendes,1 Alberto Orfao2


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Cytometry Laboratory, IPO, PORTO, Portugal Center for Cancer Research, SALAMANCA, Spain Acute leukemias of ambiguous lineage include those leukemias that show no clear evidence of differentiation along a single lineage or coexpression of markers of more than one lineage (e.g. myeloid and lymphoid). Immunophenotyping by flow cytometry is crucial for the diagnosis of these types of leukemias. Here we report a case of a 38 year old male patient, with multiple lymphadenopathies and hepato-splenomegaly and a high leukocyte count of 188x109/L. BM smears showed 80% of blast cells morphologically classified as L2 (FAB). Immunophenotyping by flow cytometry showed that blast were positive for CD7 (strong), CD13, CD34, CD56, with partial positivity for CD11b, CD33, cytCD79a and HLA-DR. They were negative for CD2, cytCD3, CD4, CD5, CD8, CD10, CD11c, CD14, CD15, CD16, CD19, CD20, cytCD22, CD36, CD64, CD65, CD117, CD123, cytIgM, Lysozyme, MPO and TdT. Complex kariotype with del (5)(q21q34) was detected. The patient achieved complete remission after AML chemotherapy and underwent an allogenic blood marrow transplantation. Flow cytometric analysis showed coexpression of lymphoid and myeloid differentiation antigens, allowing the diagnosis of acute leukemia of ambiguous lineage of mixed T/myeloid phenotype.

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POS-CC-31 THE CLINICAL AND BIOLOGICAL SIGNIFICANCE OF THE IMMUNOPHENOTYPIC ASSESSMENT OF CD81 IN MULTIPLE MYELOMA CLONAL PLASMA CELLS Bruno Paiva,1,2 Marıá-Beleń Vidriales,1,2 Norma C. Gutierrez,1,2 Marıá-Ańgeles Montalbań,3 Albert Oriol,4 Joaquıń Martıńez-Lo´pez,3 Marıá-Victoria Mateos,1,2 Lucıá Lo´pez-Corral,1,2 Jose´-J Pe´rez,1,2 Marıá-Eugenia Sarasquete,1,2 Jose´-G Laranã,5 Felipe de Arriba,6 Luis Palomera,7 Marıá-A Echebeste,8 Marıá-Jose´ Terol,9 Raquel de Paz,10 Alejandro Martin,11 Jose´ Hernańdez,12 Yolanda Gonza´lez,13 Joan Blade´,14 Juan-Jose´ Lahuerta,3 Alberto Orfao,2,15 Jesu´s-F San Miguel,1,2 on behalf of the GEM (Grupo Espanól de MM)/PETHEMA (Programa para el Estudio de la Terapeútica en Hemopatıás Malignas) cooperative study groups. 1

Hospital Universitario de Salamanca, SALAMANCA, Spain Centro de Investigacioń del Cańcer (CIC, IBMCC USALCSIC), SALAMANCA, Spain 3 Hospital 12 de Octubre, MADRID, Spain 4 Hospital Universitari Germans Trias i Pujol, Badalona; 5 Hospital Ramon y Cajal, MADRID, Spain 6 Hospital Morales Meseguer, MURCIA, Spain 7 Hospital Lozano Blesa, ZARAGOZA, Spain 8 Hospital de Donostia, SAN SEBASTIAN, Spain 9 Hospital Clıńico de Valencia, VALENCIA, Spain 10 Hospital Universitario La Paz, MADRID, Spain 11 Hospital Virgen de la Concha, ZAMORA, Spain 12 Hospital General de Segovia, SEGOVIA, Spain 13 Hospital Josep Trueta, GIRONA, Spain 14 Hospital ClUńic, IDIBAPS, BARCELONA, Spain 15 Servicio General de CitometrUá and Department of Medicine, Universidad de Salamanca, SALAMANCA, Spain 2

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The presence of CD19 in myelomatous plasma cells (MM-PC) correlates with adverse prognosis in myeloma. Although CD19 expression is regulated by CD81, this marker has been poorly investigated in myeloma. We have analyzed CD81 expression by flow cytometry in 36 smoldering (SMM) and 229 symptomatic, uniformly treated, multiple myeloma (MM) patients at diagnosis In a subset of patients (23 MM) mRNA gene expression profiling (GEP) was performed on immunomagnetically enriched MM-PC. Positive staining for CD81 was detected in MM-PC of 15/36 (42%) SMM and 90/ 229 (39%) MM patients. Interestingly, CD81þ SMM patients took a shorter time to progression (P¼.04), and CD81þ MM cases showed lower remission rates (P¼.01), progression-free (PFS; P