How many gated lymphocytes are needed for accurate assessment of T-subset percentages by flow cytometry?

Current regulatory agencies specify the use of 2,500 gated lymphocytes for accurate lymphocyte immunophenotyping by flow cytometry. However, acquisition of 2,500 gated lymphocytes is often technically infeasible when testing whole blood from lymphopenic patients. Our laboratory thus compared CD3, CD4, and CD8 percentages obtained from a lymphocyte acquisition gate of 2,500 events with those obtained, respectively, from 1,000 and 500 event acquisition gates. The study group consisted of 59 specimens with CD4 values ranging from 1% to 66%; for data analysis purposes, the group was considered as a whole and was then subdivided according to CD4 percentage (> 25%, < 25%, < 5%). For all groupings analyzed, percentages of CD3+, CD4+, and CD8+ lymphocytes were not significantly different for either 1,000-event or 500-event gates when compared to the standard 2,500 gate (paired t-test). Replicate parallel analyses of some samples indicated that comparable precision is obtained by using the alternative gates. These findings indicate that the use of smaller numbers of acquired lymphocytes is a reasonable alternative in situations where 2,500 lymphocytes cannot be attained.