Two-color flow cytometric analysis of monocyte depleted human blood lymphocyte subsets

Peripheral blood mononuclear cells (PBMC) from normal individuals were examined using 16 pairs of FITC and phycoerythrin (PE) directly conjugated monoclonal antibodies. Each pair of reagents was used to evaluate a conventional lymphocyte gate as well as open (non) gate of monocyte depleted PBMC. Parallel studies using the same panel of monoclonal antibodies were carried out on selected, nonmonocyte depleted samples. The major findings of this analysis were that 1,000-1,200 lymphocytes in a 10,000 cell analysis are found outside the lymphocyte gate and of these approximately 2/5 are CD16 positive LGL/NK cells, 2/5 are CD3 positive T cells, and 1/5 are CD19/CD20 positive B cells. Thus, it appears that 10-15% of the lymphoid cells fall outside of the conventional lymphocyte gate, and in certain settings monocyte depletion may be useful to perform more complete evaluation of the total lymphoid cell population obtained after ficoll-hypaque separation.