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

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P43. HIERARCHICAL CLUSTER ANALYSIS OF 14-PARAMETER FLOW CYTOMETRY IMMUNOPHENOTYPING IDENTIFIES SHIFTS IN THE CIRCULATING T-CELL COMPARTMENT FOLLOWING REPERFUSION IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION. Karel Fišer 1 , Jedrzej Hoffmann 2 , Jolanta Weaver 4 , Ian Dimmick 2 , Monika Loeher 2 , Hanspeter Pircher 5 , Carmen Martin-Ruiz 3 , Murugapathy Veerasamy 2 , Bernard Keavney 2 , Thomas von Zglinicki 3 , Ioakim Spyridopoulos 2 1 CLIP – Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, 2 nd Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic, karel.fiser@lfmotol.cuni.cz 2 Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom, 3 Institute of Ageing and Health, Newcastle University, Newcastle, United Kingdom, 4 Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom, 5 Department of Immunology, Institute of Medical Microbiology and Hygiene, Freiburg University, Freiburg, Germany In patients with acute ST-elevation myocardial infarction (STEMI) reopening of the infarct related coronary vessel by primary percutaneous coronary intervention (PPCI) is the most effective treatment strategy. However it bears its risks as the following myocardial reperfusion can induce myocardial injury and cell death. Therefore, future therapies for STEMI have to target causes of post-infarction heart failure, including myocardial reperfusion injury. We used 14-parameter flow cytometry immunophenotyping to investigate the involvement of T cell subtypes in reperfusion after PPCI. Using hierarchical cluster analysis (HCA) we identified a unique CD4(+)CD57(+) T-cell population in PPCI patients that reflected acute proliferation in the CD4(+) T-cell compartment. We also marked contraction of effector T cells in STEMI patients with CMV positivity, possibly contributing to development of immunosenescence in these patients. In summary, high-throughput polychromatic flow cytometry and HCA are capable of objective, time and cost efficient assessment of the individual T-cell immune profile in different stages of coronary heart disease and have broad applications in clinical trials. Acknowledgements This work was supported by University research centre (UNCE) grant, UNCE 204012. Analytical Cytometry VII 139
P44. INSTRUMENT SETTINGS FOR EUROFLOW STANDARDIZED 8-COLOR PANELS ON DIFFERENT FLOW CYTOMETRY PLATFORMS Michaela Nováková 1 , Marcela Vlková 2 , Daniel Thürner 1 , Juan Flores Montero 3 , Mikael Roussel 4 , Ana Helena Santos 5 , Ester Mejstříková 1 , Quentin Lecrevisse 3 , Ondřej Hrušák 1 , Tomáš Kalina 1 1 Pediatric Hematology and Oncology, Charles University Prague, 2 nd Medical Faculty, Prague 5, Czech Republic, michaela_novakova@lfmotol.cuni.cz 2 Department of Clinical Immunology and Allergology, St. Anne’s University Hospital and Faculty of Medicine, Masaryk University, Brno, Czech Republic 3 Cancer Research Center (IBMCC-CSIC), Department of Medicine and Cytometry Service, University of Salamanca, Salamanca, Spain 4 Hematology Laboratory, CHU Pontchaillou, Rennes, France 5 Centro Hospitalar do Porto, Portugal Background: EuroFlow consortium has recently developed a standardized approach to immunophenotyping of hematological malignancies (Kalina et al., 2012; van Dongen et al., 2012). The standardization is performed on both levels, uniform antibody panels and uniform instrument settings, so that a computational meta-analysis of the data is possible. However, due to availability of the instruments at the project’s beginning in 2006, we have proven the approach only on 8-color BD Biosciences digital instruments. Lately, 8-color flow cytometry has become available on several flow cytometry platforms of different makers. Thus, we tested the feasibility of standardized acquisition and merged analysis of data across the platforms. Methods: We have set the PMT voltage using hard-dyed 8-peak Rainbow beads to reach common target channel values. Where needed, target values were re-scaled to 18-bit common scale. Since the fluorochromes used in Rainbow beads do not completely correspond to fluorochromes used in Lymphocytosis Screening Tube, we used 2 types of capture beads stained with actual monoclonal antibodies to achieve more accurate target values for different instruments. We have stained peripheral blood of three healthy donors with modified EuroFlow Lymphocytosis Screening Tube to obtain discrete positive lymphocyte subset in all 8 channels. After staining we split the samples and acquired on BD FACS Canto II, BC Navios, BC Cyan ADP and Miltenyi MACSQuant Analyzer. Next, we re-scaled to 18-bit, merged and analyzed in Infinicyt software. Improved target values were confirmed by measuring three peripheral blood samples from healthy donors in 5 Euroflow centers (Prague, Brno, Salamanca, Rennes, Porto) on 3 types of instruments (5 BC Navios, 3 BD Canto II, 3 Dako Cyan). Results: Data obtained from the same sample on different cytometry platforms were very similar. After gating lymphocytes on FSC and SSC (scatter parameters were not standardized), we could apply the same gating strategy (gate position) on all samples. When we analyzed MFI of gated positive subsets, the values were distributed with average CV of 18,8% (range 7% - 32%), which presents variability that is lower than both the inter-individual and inter-laboratory variability based on the previous quality control 140 Analytical Cytometry VII
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ABSTRACTS - ORAL PRESENTATIONS 14.
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and (iii) siRNA-mediated knockdown
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25. PROFILING OF CHILDHOOD ACUTE LE
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more dominantly than Th1 and CD4 ce
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29. INFLUENCE OF THE LEVEL OF CD133
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36. B-CELL IMMUNOPHENOTYPING OF LAR
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possible mechanism behind risk alle
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isotype controls and FMO for CD14 a
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NEP developmental stages were disti
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50. THE PROGRESSION OF HIV INFECTIO
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52. PRŮKAZ REGULAČNÍCH T LYMFOCY
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values when analysing paediatric ly
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42,0) vs. 1,5 (0,0-28), p
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for application of proton therapy,
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effects will be also characterized
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Although multiple signalling pathwa
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an ability to recreate the tumour f
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incubated with non-filtered superna
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approaches how to detect and isolat
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progress in development of automate
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tissues is characteristic for a num
Page 43 and 44: kappaB. Beside that, we found OANO
Page 45 and 46: therapy in patients (Calderwood et
Page 47 and 48: difference in the effect caused eit
Page 49 and 50: 4 CIC bioGUNE Technology Park of Bi
Page 51 and 52: P8. PROADIFEN (SKF-525A) ATTENUATES
Page 53 and 54: models. As manifested by compromise
Page 55 and 56: P11. NEW BIOACTIVE AND FLUORESCENT
Page 57 and 58: Acetylation of histones, along with
Page 59 and 60: stimulated cells. The physiological
Page 61 and 62: h induced very minor manifestations
Page 63 and 64: chemotherapeutic drug LA-12. These
Page 65 and 66: aberrant expression, localization a
Page 67 and 68: P21. ASSESSMENT OF MITOCHONDRIAL FU
Page 69 and 70: P23. PTEROSTILBENE: COMPARISON OF A
Page 71 and 72: Fam123b. Amer1 has been shown very
Page 73 and 74: In summary, we described different
Page 75 and 76: this process. Combined treatment wi
Page 77 and 78: Homolog 1) gene; previously identif
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Page 81 and 82: was harmful neither alone nor in co
Page 83 and 84: P34. PHARMACOLOGICAL INHIBITION OF
Page 85 and 86: novel chemotherapeutics with specif
Page 87 and 88: P38. EVIDENCE OF ABNORMAL PERIPHERA
Page 89 and 90: acid to 5-lipoxygenase. Similarly t
Page 91 and 92: P41. WHY CAN WE SEE DIFFERENT RADIO
Page 93: P42. ELUCIDATION OF CROSSTALK BETWE
Page 97 and 98: e used to cultivate endothelial cel
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Page 101 and 102: P49. NEW ANALOGS OF RHODAMINE Jiři
Page 103 and 104: P51. FLUORESCENCE-LABELED FERROMAGN
Page 105 and 106: could be achieved via the activatio
Page 107 and 108: 28. Pp. 1565-1570. 2008. Dearden C:
Page 109 and 110: cells (SFC) using IFN-γ Elispot in
Page 111 and 112: FACSCantoII flow cytometer (Becton
Page 113 and 114: P59. EARLY DIAGNOSTICS OF CARTILAGE
Page 115 and 116: cross-reacting group (CREG) and sha
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Page 119 and 120: non-covalent bonds. For a solution
Page 121 and 122: P65. OVULATION STIMULATION PROTOCOL
Page 123 and 124: Acknowledgements This work was supp
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Page 131 and 132: P73. THE V-ATPASE-INHIBITOR BAFILOM
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Page 135 and 136: We conclude that decrease in the po
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Page 141 and 142: LD11015, from GAČR 13-29358S, and
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given cell type are missing and the
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and HistoPARK (CZ.1.07/2.3.00/20.01
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Our results show that both p38α +/
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P90. TOLL-LIKE RECEPTOR 2 TRACKS TH
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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.