A flow cytometric method for enumeration of lymphocyte sub-populations in sample containing lysis-resistant red blood cells.
Determination of lymphocyte sub-populations is usually carried out by flow cytometry using two-color immunophenotyping reagent. By this technique, however, the combination of FSC and SSC with CD45-FITC/CD14-PE is unable to identify the lymphocyte population in a sample containing lysis-resistant red blood cells (RBC). The actual values of lymphocyte sub-populations, therefore, cannot be determined in these RBC contaminated samples. To overcome this problem, we describe here the use of 7-aminoactinomycin D (7-AAD) to exclude lysis-resistant RBC from white blood cells (WBC). By adding 7-AAD, lymphocytes of samples containing RBC could be identified by using FL3/SSC, therefore, the actual number of lymphocyte sub-populations of the stained cells was obtained. We have proved that 7-AAD can be used to exclude contaminated RBC and has no effect on the measurement of lymphocyte sub-populations by using two-color immunophenotyping reagent. In routine blood samples that contain lysis-resistant RBC, 7-AAD markedly increased the purity of lymphocytes in the lymphocyte gate to >95% and the lymphocyte sub-populations therefore could be correctly determined. The described method is inexpensive, simple and gives successful analysis of lymphocyte sub-populations in a sample containing lysis-resistant RBC.
Kasinrerk
Watchara
W
Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. watchara@chiangmai.ac.th
D000818:Animals; D017252:CD3 Complex / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D015658:HIV Infections / Q000097:blood; D006461:Hemolysis; D006801:Humans; D016130:Immunophenotyping; D016131:Lymphocyte Subsets / Q000166:cytology*
D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D003609:Dactinomycin; C025942:7-aminoactinomycin D