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

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