Open AccessComparative analysis of whole blood lysis methods for flow cytometry
Author(s) -
Bossuyt Xavier,
Marti Gerald E.,
Fleisher Thomas A.
Publication year - 1997
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19970615)30:3<124::aid-cyto3>3.0.co;2-l
Subject(s) - flow cytometry , lysis , immunophenotyping , cytometry , microbiology and biotechnology , cd8 , biology , cd3 , staining , whole blood , chemistry , immunology , pathology , medicine , antigen
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 for ≈80% of all events with ACK and Ortho‐mune. The other lysing methods consistently yielded less debris (≈50%) with Immunolyse generating only ≈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 determinations 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. Cytometry 30:124–133, 1997. © 1997 Wiley‐Liss, Inc.