A new method for measuring lymphoproliferation at the single-cell level in whole blood cultures by flow cytometry

A simple and reproducible method is described for the measurement of proliferative responses to mitogens and antigens. Whole blood (WB) cultures are used and separation procedures are not necessary. Proliferative responses are monitored as development of lymphoblasts determined by flow cytometry (FC). Lymphocytes and lymphoblasts were shown to be exclusively selectable from mixed cell populations of WB by their light scatter profile on FC analysis. More than 90% of the lymphoblasts identified by FC were replicating DNA as determined by incorporation of the thymidine analogue 2-bromo-5-deoxyuridine. The WB/FC technique can be combined with triple immunofluorescence which was illustrated by determining subsets of lymphoblasts developing following stimulation with various mitogens and antigens. The new method was compared with the conventional method (peripheral blood mononuclear cell culture and proliferation measured by detection of DNA synthesis). The reproducibility of the methods were not different. The antigen-induced responses recorded by both methods correlated, but with significantly higher stimulation indices obtained by WB/FC. The latter procedure was also technically easier to perform and less time-consuming.