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[Immunophenotyping of lymphocyte subpopulations using monoclonal antibodies in an analytical flow cytometer:
Abstract:
Analytical flow cytometers which are easy to operate, and monoclonal antibodies specific for cell surface determinants of lymphocytes, offer a simple and rapid method for the identification of the immunological phenotype of lymphocyte subsets. We investigated mononuclear cells isolated from EDTA-blood bv density gradient centrifugation. The intraassay precision (n = 12) of the determination by means of monoclonal antibodies directed against T-cells (Leu 4), T-helper/inducer-cells (Leu 3 a, b), T-suppressor/cytotoxic cells (Leu 2 a) and cells of the 'NK'-phenotype (Leu 7) varied between 2.2 and 7.9%. Studies using the subset markers Leu 2a and Leu 3a, b conjugated either to fluorescein isothiocyanate (FITC) or to phycoerythrin (PE) showed comparable results for both subsets. Dual immunofluorescent analysis using two different antibodies conjugated to contrasting fluorochromes resulted in slightly higher fractions of T-helper/inducer-cells (Leu 3a, b-FITC: a = 0.97, b = 3.18, r = 0.98) and T-suppressor/cytotoxic cells (Leu 2 a-PE: a = 0.99, b = 2.22, r = 0.97) compared with single immunofluorescent analysis. We found no constant association to explain this statistically significant discrepancy (p less than or equal to 0.0002). The investigation of 100 controls established the following reference values for the fraction of lymphocyte subsets (median, 95%-interval): T-cells 0.75 (0.57-0.87), T-helper/inducer-cells 0.47 (0.34-0.63), T-suppressor/cytotoxic cells 0.26 (0.15-0.42), 'NK'-cells 0.16 (0.04-0.34). The corresponding absolute numbers of cells are: T-cells 1.300 (0.730-2.240), T-helper/inducer-cells 0.820 (0.44-1.540), T-suppressor/cytotoxic cells 0.450 (0.190-0.915), 'NK'-cells 0.300 (0.060-0.670).