Comparative analysis of whole blood lysis methods for flow cytometry.
We performed a parallel evaluation of six whole blood lysis methods comparing light scatter and quantitative fluorescence intensity based on quantitative flow cytometry, of selected lymphocyte subsets and CD34+ cells. Leukocytes prepared with FACS Lysing Solution (BDIS), Immunolyse (Coulter) and Optilyse B (Immunotech) consistently gave lower forward scatter values than those prepared with ACK (BioWhitaker), Ortho-mune (Ortho) and ImmunoPrep (Coulter). Debris, defined as CD45 negative events with the threshold off, accounted approximately 80% of all events with ACK and Ortho-mune. The other lysing methods consistently yielded less debris (approximately 50%) with Immunolyse generating only approximately 16% debris. Optilyse and FACS lyse consistently displayed the lowest percentage of lymphoid cells (CD45+/CD14-) in the three part differential. The percentage of CD3+, CD20+, CD5+, and CD16/CD56+ cells was consistent with all methods but CD4 and CD8 determinants showed inconsistent variation with ACK and Ortho-mune. In addition, the fluorescence intensity of CD14 PE and CD8 PE staining was markedly decreased on cells prepared with ImmunoPrep. Finally, the clearest separation of CD34+ cells was observed with ACK and Ortho-mune. Our data demonstrate that the method used for red cell lysis can have definite impact on immunophenotyping and selected methods appear to be more suitable for specific applications.
Bossuyt
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Immunology Service, National Institutes of Health, Bethesda, MD 20892-1508, USA.
Marti
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Fleisher
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D000911:Antibodies, Monoclonal; D018952:Antigens, CD34 / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007202:Indicators and Reagents; D007962:Leukocytes / Q000166:cytology* / Q000276:immunology; D016131:Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D000954:Antigens, Surface; D005456:Fluorescent Dyes; D007202:Indicators and Reagents