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

• Published in: Journal of immunological methods Vol. 195; no. 1; pp. 63 - 72
• Main Authors: Gaines, Hans, Andersson, Lena, Biberfeld, Gunnel
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 09.09.1996; Elsevier
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; Cell Culture Techniques - methods; Cell Division; Flow cytometry; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Humans; Lymphocytes - cytology; Lymphoproliferation; Male; Medicin och hälsovetenskap; Molecular and cellular biology; Whole blood; TT; PBS; Flow cytometry; PBMC DNA; cpm; PHA-1; PHA-p; PPD; PWM; WB; PBMC; CMV; Whole blood; VZV; Lymphoproliferation; WB FC; FC; BrdU; ConA; Performance evaluation; Human; Cell proliferation; Lymphoblast; Measurement method; Blood culture; Lymphocyte
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(96)00085-3

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.