QUANTITATIVE-DETERMINATION OF SURFACE ANTIBODY-BINDING CAPACITIES OF IMMUNE SUBSETS PRESENT IN PERIPHERAL-BLOOD OF HEALTHY ADULT DONORS

The objective of this study was to quantitate the antibody binding cap acity (ABC) of CD3, CD4, CD8, CD16, and CD19 on lymphocytes and CD4 on monocytes from healthy adult donors. Peripheral blood was collected o ver three consecutive days and repeated in the same format two weeks l ater for comparison to initial measurements. Immune subsets were label ed by direct single or two-color staining in whole blood followed by l ysis of erythrocytes. Fluorescence intensity measurements were made by carefully calibrating the flow cytometer and then measuring the inten sity of monoclonal antibody staining on labeled cells and on Quantum S imply Cellular Microbeads. The effect of paraformaldehyde fixation on intensity measurements and coefficient of variation of thirty replicat es for each phenotype were also studied. We found a small change in ca lculated ABC following overnight fixation with a greater change follow ing 48 h of fixation prior to flow cytometric analysis. We found excel lent precision could be achieved for measuring the ABC of most markers with some improvement desirable for expression of CD4 on monocytes an d CD16(+) lymphocytes. Between donors we found a high-law range of CD3 (+) = 134,34-45,905; CD4(+) (lymphocytes) = 54,174-36,106; CD4(+)(mono cytes) = 9,246-3094; CD8(+) = 268,868-190,622; CD3(+)CD8(+) = 269,858- 212,024; CD16(+) = 38,307-336; and CD19(+) = 25,252-11,689. For the to tal donor group, the observations at week 1 and week 2 were not signif icantly different (alpha = .05) for any of the immunophenotypes we stu died. The data presented here continue to show that it is possible to perform quantitative intensity measurements of immune subsets when per forming immunophenotyping studies. (C) 1996 Wiley-Liss, Inc.