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.