Lymphocyte subset analysis by Boolean algebra: a phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence

Abstract

Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated 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-), and 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 antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.