Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.

Cancer Research Center CB16 12 00400 Instituto de Salud Carlos 3 Madrid Spain

CLIP Cytometry Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University Prague Czech Republic

Cytognos SL Salamanca Spain

Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden the Netherlands

Department of Immunology Erasmus MC Erasmus University Medical Center Rotterdam the Netherlands

Department of Pediatric Hematology and Oncology Medical University of Silesia in Katowice Zabrze Poland

FACS Stem cell Laboratory Institute of Laboratory Medicine Kantonsspital Aarau AG Aarau Switzerland

Rostock University Medical Center Division of Internal Medicine Medical Clinic 3 Hematology Oncology and Palliative Medicine Special Hematology Laboratory Rostock Germany

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