A rapid technique for lymphocyte preparation prior to two-color immunofluorescence analysis of lymphocyte subsets using flow cytometry Comparison with density gradient separation

Abstract

A technique is described for lymphocyte preparation which permits analyses by two-color immunofluorescence and flow cytometry. It consists, briefly, of the lysis of red blood cells and washing of white blood cells prior to labeling. We tested this technique with a large panel of monoclonal antibodies in mono- and dual immunofluorescence. By comparing these results to those obtained after density gradient separation, we found the following statistically significant differences: the count of the phenotype B1 + was higher after whole blood lysis preparation than after density gradient separation; whereas, the corresponding counts of OKT4 + and Leu-4 −Leu-7 + phenotypes were lower. No difference was detected with OKT8 +, Leu-4 +, OKT8 +Leu-4 +, OKT8 +Leu-4 −, OKT8 −Leu-4 +, OKT8 +Leu-7 +, Leu-4 +Leu-7 +, Leu-4 −Leu-11c + OKT8 +Leu-11c + and OKT8 +Leu-15 + phenotypes. We have studied the reproducibility of both methods and the correlation between them. The disparity of the lymphocyte subset count between these two methods, though statistically significant, was relatively weak and seems to be due to the density gradient separation. Since the preparation of lymphocytes using the density gradient method is time consuming, we propose whole blood lysis as an alternative lymphocyte separation method when assessing immune status in human disease by flow cytometry. It offers the following advantages: (i) it does not require additional steps, (ii) it permits two-color immunofluorescence through the labeling of white blood cells after washing, (iii) it is reliable, (iv) it is reproducible, and (v) it is helpful in studies of lymphopenia since it offers the possibility of lymphocyte enrichment.