LYMPHOCYTE SUBSET ANALYSIS BY BOOLEAN-ALGEBRA - A PHENOTYPIC APPROACHUSING A COCKTAIL OF 5 ANTIBODIES AND 3-COLOR IMMUNOFLUORESCENCE

Commercial reagent kits for the evaluation of leukocyte subsets involv e the staining of a panel of up to six tubes using combinations of pre -mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conj ugated monoclonal antibodies. We describe a rapid method whereby total CD3(+) T-cells, CD4(+) T-cells (CD3(+)CD4(+)), CD8(+) T-cells (CD3(+) CD8(+)), putative gamma delta-receptor-T-cells (CD3(+)CD4(-)CD8(-)), a nd T-cells that are CD3(+)CD4(+)CD8(+) as well as B-lymphocytes and NK -cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated a ntibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, a nd either peridinin chlorophyll protein (PerCP) or allophycocyanin (AP C) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Bec ton Dickinson, San Jose, CA) and all of the lymphocyte subset values w ere determined by Boolean algebra using a technique we refer to as Boo lean gate analysis (BGA). Our study has shown that BGA is statisticall y equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequenti al saving in reagents, consumables, and time. (C) 1994 Wiley-Liss, Inc .