[Effect of plasma on analysis of lymphocyte subsets]

We examined the effect of plasma on the analysis of lymphocyte subsets with a flow cytometer using whole blood cells. Removal of plasma from whole blood by washing them before labelling the lymphocytes with fluorescein-conjugated antibodies reduced the proportion of CD5+CD19+ cells and increased the proportions of CD16+ CD57- and CD16+ CD57+ cells, as compared with those measured without washing, but did not change the proportions of CD5+ CD19-, CD5- CD19+, CD4+ CD8-, CD4- CD8+ and CD16- CD57+ cells. Removal of plasma from whole blood also reduced the fluorescence intensity of CD4 and CD5 antigens and increased that of CD8, CD16, CD19 and CD57 antigens on each lymphocyte subsets. Characteristics of the changed lymphocyte subsets were to have a surface antigen with weak immunofluorescence on the flow cytometric analysis such as CD5 and CD16, and to have an unclear borderline between the positive and negative cells. Therefore, even slight changes in the fluorescence intensity of these antigens could change the proportion of CD5+ CD19+, CD16+ CD57- and CD16+ CD57+ cells. However, these changes were not observed, when using the washed blood cells as samples after readdition of plasma to them. These data suggest that removal of plasma from blood before labelling the lymphocytes with fluorescein-conjugated antibodies is necessary to make the sample condition equal for flow cytometric analysis of lymphocyte subsets.