WHOLE-BLOOD METHOD FOR SIMULTANEOUS DETECTION OF SURFACE AND CYTOPLASMIC ANTIGENS BY FLOW-CYTOMETRY

Dual parameter analysis of surface antigens in flow cytometry has beco me a standard method for detection of cell subsets. However, few metho ds have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a sim ple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells a re lysed with 2% acetic acid. Preparation of samples in this manner re sulted in altered light scatter characteristics relative to unpermeabi lized samples; however, gating issues were overcome using a combinatio n of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD 3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure a nd fluorescence vs. scatter gating. The effectiveness of the permeabil ization process was assessed using a monoclonal antibody designated TI A-1, which is specific for a cytoplasmic antigen associated with cytot oxic granules predominantly found in CD8+ cells. The method effectivel y quantitated TIA-1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simpl ified procedure for simultaneous identification of surface and cytopla smic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system.