TECHNICAL INFLUENCES ON IMMUNOPHENOTYPING BY FLOW-CYTOMETRY - THE EFFECT OF TIME AND TEMPERATURE OF STORAGE ON THE VIABILITY OF LYMPHOCYTE SUBSETS

The typing of lymphocyte subsets may be influenced by a variety of technical influences including the duration and temperature of sample storage and the method used for staining samples. We have extended a previous study examining the effect of storage conditions on the baseline values of a number of lymphocyte subsets. EDTA-anticoagulated samples from 13 HIV-1-positive and 15 healthy laboratory controls were analyzed for a number of lymphocyte subsets (CD3+, CD4+/CD3+, and CD8+/CD3+ T cells and CD19+ B cells) (whole blood lysis method, Becton-Dickinson FACScan flow cytometer and reagents) at 0, 24, 48, 72, and 96 h after storage at 4-degrees-C, 17-degrees-C or 21-degrees-C. During storage at both 4-degrees-C and 21-degrees-C, there were significant changes in baseline values of the majority of lymphocyte subsets and some of these were related to the HIV status of the donor. ne optimum temperature for storage in our system appeared to be around 17-degrees-C in both our study groups. We have also used propidium iodide in order to discriminate between viable and non-viable cells during flow cytometry of lymphocytes from eight HIV-1-positive and five control subjects. The results show that for both HIV-positive and control samples stored at 4-degrees-C, and for control subjects at 21-degrees-C, the changes in baseline values of lymphocyte subsets observed were not due to selective loss of particular subsets arising from cell death during storage. However, there was substantial loss of cells from all three subsets in HIV-positive subjects during storage at 21-degrees-C, with loss of CD8+ and CD3+ T cells being more significant than loss of CD4+ T cells.