PLOW CYTOMETRIC IMMUNOPHENOTYPING OF LYMPHOCYTE SUBSETS IN SAMPLES THAT CONTAIN A HIGH PROPORTION OF NONLYMPHOID CELLS

Flow cytometric (FCM) immunophenotyping of peripheral blood from thala ssemia patients presents technical difficulties because of the high pr oportion of immature red cells, The combination of forward scatter (FS C) and side scatter (SSC) with fluorescence associated with human leuk ocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was neces sary to exclude immature red cells to analyze lymphocyte subsets in th ese patients, A simultaneous three-color FCM method was developed, wit h the basis that transferrin receptor (CD71) or glycophorin A (glyco A ) is present on all immature red cells, but is not expressed on CD45 p ositive leukocytes. In this study, the lymphocyte population was ident ified by gating out unwanted cell populations using the FSC/ CD71-fluo rescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, g lyco A-FITC negative cells, or CD45-PerCP positive cells were identifi ed, then analyzed on the basis of FSC/SSC to allow any remaining non-l ymphocyte cells in FSC/SSC gate to be excluded, The cells in FSC/SSC g ate were then analyzed using other irrelevant two-color antibodies, Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method, Both methods markedly increas e the purity of lymphocytes in the analysis gate, Either method is eas y, straightforward, requires a six-tube set of reagent tubes, and prov ides a reliable method for immunophenotyping lymphocyte subsets in pre parations containing a large percentage of nonlymphoid cells, (C) 1994 Wiley-Liss, Inc.