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 capacity (ABC) of CD3, CD4, CD8, CD16, and CD19 on lymphocytes and CD4 on monocytes from healthy adult donors. Peripheral blood was collected over three consecutive days and repeated in the same format two weeks later for comparison to initial measurements. Immune subsets were labeled by direct single or two-color staining in whole blood followed by lysis of erythrocytes. Fluorescence intensity measurements were made by carefully calibrating the flow cytometer and then measuring the intensity of monoclonal antibody staining on labeled cells and on Quantum Simply Cellular Microbeads. The effect of paraformaldehyde fixation on intensity measurements and coefficient of variation of thirty replicates for each phenotype were also studied. We found a small change in calculated ABC following overnight fixation with a greater change following 48 h of fixation prior to flow cytometric analysis. We found excellent precision could be achieved for measuring the ABC of most markers with some improvement desirable for expression of CD4 on monocytes and CD16+ lymphocytes. Between donors we found a high-low range of CD3+ = 134,349-45,905; CD4+ (lymphocytes) = 54,174-36,106; CD4+ (monocytes) = 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 total donor group, the observations at week 1 and week 2 were not significantly different (alpha = .05) for any of the immunophenotypes we studied. The data presented here continue to show that it is possible to perform quantitative intensity measurements of immune subsets when performing immunophenotyping studies.

Evaluation of T-lymphocyte subsets present in semen and peripheral blood of healthy donors: a report from the heterosexual transmission study.

The purpose of this study was to accurately determine the T-lymphocyte subsets found in semen from healthy volunteers, to evaluate the impact of repeated ejaculation on the frequency or type of immune cells present in semen, and to compare subset analysis in semen to that in the peripheral blood. To accomplish this, a flow cytometric method was developed to identify and count immunophenotypically distinct cells present in semen. Fresh semen samples and peripheral blood were collected over three consecutive days from nine healthy donors. Donors had normal ejaculate volume, sperm count, sperm motility, morphology, and leukocyte count. No significant intra-donor differences were seen in these parameters over time. No significant differences were observed in the percentage of CD3+ cells, CD4+ cells, CD8+ cells, and the CD4:CD8 ratio in semen on consecutive days. However, within the CD4+ subset, when naive and memory CD4+ cells were measured, some day to day variability was suggested. No significant differences in CD3+, CD4+, CD8+, CD4/CD8 ratio, or naive and memory subsets were seen in the peripheral blood between sampling days. When semen was compared to peripheral blood some differences in immune subset values were observed, with an increase in the percentage of memory CD4+ cells in semen being the most striking. This finding may be relevant to HIV transmission, since others have shown that this cell may be preferentially infected with HIV and is the primary reservoir for virus in infected individuals.