ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.

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al., Adhesion molecule expression on peripheral blood mononuclear cells in rheumatoid arthritis: positive correlation between the proportion of L-selectin and disease activity. Clin Rheumatol, 1995. 14(3): p. 335-41. 7. Tanaka, M., et al., Expansion of a unique macrophage subset in rheumatoid arthritis synovial lining layer. Clin Exp Immunol, 2008. 154(1): p. 38-47. 8. Stuhlmuller, B., et al., CD11c as a transcriptional biomarker to predict response to anti-TNF monotherapy with adalimumab in patients with rheumatoid arthritis. Clin Pharmacol Ther, 2010. 87(3): p. 311-21. 41. IMMUNOPHENOTYPE ANALYSIS OF NUCLEATED ERYTHROID PROGENITORS. APPLICATION OF OBTAINED RESULTS IN LEUKAEMIA DIAGNOSTICS Michaela Fajtová 1,2 , Anna Kovariková 1,2 , Jan Sedlák 1 1 Cancer Research Institute 2 Centre for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovak Republic; e-mail: michaela.fajtova@savba.sk Introduction: Nucleated erythroid progenitors (NEPs) in normal regenerating bone marrow (BM) comprised of 4 developmental stages – pro-erythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. In clinical laboratories, the erythroid phenotype is conventionally characterised by the expression of CD36, CD71, and CD235a antigens, however the analysis of these antigens is not sufficient for the distinction of NEPs into 4 developmental stages. We supplemented previous NEP phenotype analysis (Wood, 2004) with the analysis of additional markers on NEPs, including CD105, CD117, CD45, CD38, we proposed NEPs gating strategy and determined the percentage of NEPs developmental stages in regenerating BM (Fajtova et al., 2013). The knowledge of exact erythroid phenotype is helpful for uncovering neoplastic erythroid population in diagnosis and follow-up of acute erythroid leukaemia (AML-M6) and myelodysplastic syndrome (MDS). Material and methods: NEPs phenotype was analysed on normal BM samples from 6 non-leukemic and 14 leukemic patients (mainly B-ALL) in complete remission. Aberrant NEPs phenotype was analysed on BM samples from AML-M6 patients in diagnosis and follow-up. Flow cytometric measurement was performed on a BDCantoII Flow Cytometer using 6-colour staining. NEPs phenotype profile was examined with antibodies: anti- CD36-FITC, anti-CD36-FITC/CD235a-PE, anti-CD71-PE, anti-CD105-PerCP-Cy5.5, anti- CD34-PE-Cy7, anti-CD117-APC, anti-HLA-DR-FITC, anti-CD38-PE, anti-CD45-VioGreen and nucleic dye Syto. Results: All NEP developmental stages were gated as cells with the highest CD71 intensity and were stained with the nucleic acid dye Syto. Alternatively, NEPs in all stages could be also gated as CD36 + CD45 -/low Syto + cells, as CD45 -/low gating excluded the monocytic population and Syto + gating excluded the denucleated erythro debris (CD36 + Syto - ). CD36 + CD45 -/low Syto + gating is necessary in samples lacking CD71 antigen expression on NEP population. Analytical Cytometry VII 61
NEP developmental stages were distinguished on the basis of different values of CD105, CD117, FSC parameters. Pro-erythroblasts were characterized by the expression of CD71 + , CD105 + , CD117 + and the highest value of FSC; basophilic erythroblasts by the expression of CD71 + , CD105 + and decreased FSC; polychromatophilic erythroblasts by the expression of CD71 + and medium FSC; orthochromatophilic erythroblasts by the CD71 + and the lowest FSC value. NEP developmental stages expressed CD36, CD38 and CD45 in various intensity and did not express CD34 and HLA-DR. Knowledge of the expression profiles of normal NEPs, we used for the analysis of erythroid population in two cases of AML-M6 subtype erythroleukaemia, which we were analysed in the years 2008-2013. In first case of erythroleukaemia we identified the presence of myeloblasts (9% from BM nucleated cells) with aberrant phenotype (CD7 + , CD22 + ) and NEPs (75%). NEPs comprised of amplified cells of different developmental stages (7% pro-erythroblasts, 5.4% basophilic erythroblasts, 21.5% polychromatophilic erythroblasts, and 38.8% orthochromatophilic erythroblasts). All stages displayed aberrantly reduced CD71 expression (decreased intensity) and orthochromatophilic erythroblast expressed slightly decreased intensity of CD36 (90%). Patient underwent treatment, achieved complete remission and subsequently underwent allogeneic stem cell transplantation. Nine months after diagnosis, this patient relapsed, showing the presence of myeloblasts (21%) and NEPs (49%) in the BM. Pro-erythroblasts and basophilic erythroblasts possessed normal phenotype, polychromatophilic erythroblasts displayed aberrantly reduced CD71 expression (50%), and orthochromatophilic erythroblasts lost CD71 expression (1%) and showed reduced CD235a expression (30%). In second case of erythroleukaemia we identified in diagnosis the presence of myeloblasts (6%) with aberrant phenotype (CD38 dim , CD15 dim , CD13 - ) and NEPs (82%) in the BM. Neoplastic NEPs comprised of mass of cells with aberrant phenotype (CD36 neg-bright , CD71 medium , CD235a + , CD105 dim , HLA-DR dim , CD38 dim a CD117 - ), aberrantly increased SSC characteristic and without the possibility to distinguish different NEPs developmental stages. Detection of minimal residual disease by flow cytometry on day 33 and 64 was based on identification of residual NEPs with aberrant phenotype. However, during follow-up we identified NEPs (2%), in which it was possible to identify all 4 developmental stages with normal phenotype. Conclusions: AML-M6 is very rare type of AML that accounts for less than 5% of adult AML cases. MDS is more frequent clonal malignant disorder, in which also NEPs could possess phenotypic abnormalities, such as asynchronous expression of CD71 versus CD235a (Malcovati et al., 2005), altered distribution of nucleated red blood progenitors and increased numbers of CD36 -/low NEPs (Matarraz et al., 2010). In clinical laboratories, the phenotype of neoplastic NEPs in AML-M6 or MDS is evaluated as a one whole that causes losing information about aberrant phenotypes on different NEP developmental stages - important information for leukaemia follow-up. Our proposed NEP gating scheme allows for the exact assessment of phenotype of different NEP developmental stages in BM. Evaluation of CD71, CD235a, CD117, CD105, CD36, CD45 antigen expression is also recommended in standardized Euroflow panels for AML/MDS. 62 Analytical Cytometry VII
Page 1 and 2: ABSTRACTS - ORAL PRESENTATIONS 14.
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P21. ASSESSMENT OF MITOCHONDRIAL FU
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P23. PTEROSTILBENE: COMPARISON OF A
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In summary, we described different
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Homolog 1) gene; previously identif
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was harmful neither alone nor in co
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P34. PHARMACOLOGICAL INHIBITION OF
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novel chemotherapeutics with specif
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P38. EVIDENCE OF ABNORMAL PERIPHERA
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acid to 5-lipoxygenase. Similarly t
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P41. WHY CAN WE SEE DIFFERENT RADIO
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P42. ELUCIDATION OF CROSSTALK BETWE
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P44. INSTRUMENT SETTINGS FOR EUROFL
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cells (SFC) using IFN-γ Elispot in
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FACSCantoII flow cytometer (Becton
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P59. EARLY DIAGNOSTICS OF CARTILAGE
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cross-reacting group (CREG) and sha
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the frequency of autoimmune disease
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non-covalent bonds. For a solution
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P65. OVULATION STIMULATION PROTOCOL
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Acknowledgements This work was supp
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triggers a process of normal blood
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P75. 5-FLUOROURACIL-SELECTED TUMOUR
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We conclude that decrease in the po
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Acknowledgements This work was supp
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Our results show that both p38α +/
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for chemokines in attracting “inf
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in immune organs (Bursa of Fabricii
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ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.