Flow cytometric lymphocyte subset analysis using material from frozen whole blood

Multicenter immune monitoring programs commonly rely on storing and shipping cryopreserved peripheral blood mononuclear cells (PBMC), isolated from whole blood before freezing. However, under many conditions in the field, facilities to separate PBMC are absent. Here, we investigate the feasibility of using whole blood (WB) frozen at -80°C as a source of viable lymphocytes for use in immunological studies. We compare the percentage of CD4 and CD8 T lymphocytes and their subsets from frozen WB with results from cryopreserved PBMC in five random healthy blood donors (three female, two male). We report that CD4 and CD8 values in lymphocytes from WB frozen up to 120 days were very similar to those of PBMC frozen up to 10 days. These data suggest that within the limits of parameters investigated in this study, contrary to our original assumptions, whole blood frozen at -80°C may in fact be an appropriate source of viable lymphocytes for T cell enumeration assays in immunological and epidemiological studies.