Analysis of T cell subsets in normal adults. Comparison of whole blood lysis technique to Ficoll-Hypaque separation by flow cytometry

Abstract

Analysis of peripheral blood T cells and T cell subsets by immunofluorescence and flow cytometry is employed commonly in studies of normal individuals and in certain disease states. Methods for separating T cells from granulocytes and erythrocytes vary. We have sought to determine whether or not such procedures alter the composition of cells being analyzed. In this communication, we report that, in normal individuals, Ficoll-Hypaque separation of lymphocytes selectively decreases the OKT8+ subset compared to the whole blood lysis technique (P less than 0.01). The decrease in the OKT8+ subset results in a significant increase (P less than 0.01) in the OKT4/OKT8 ratio. This disparity appears to be due to a selective loss of OKT8+ cells during centrifugation over Ficoll-Hypaque. With the Ficoll-Hypaque method, we have also found that the fixation of lymphocytes reacted with monoclonal antibodies is less stable than cells prepared by the whole blood lysis method. The interpretation of measurements of T cell subsets is influenced by the method by which these cells are isolated.