Comparison of two flow cytometric methods enumerating CD4 T cells and CD8 T cells

The enumeration of blood CD4(+) (single-positive)T cells by flow cytometry is subject to errors such as counting CD4(+) monocytes or CD4(+)CD8(+) (double-positive) T cells as CD4(+) T cells. Relatively accurate count can be obtained when CD4(+) T cells are defined as CD3(+)CD4(+)CD8(-) mononuclear cells (MNCs), using 3-color flow cytometry. However, using this approach, further classification into CD4+ T cell subsets requires expensive 4-color now cytometry, In an attempt to enumerate CD4(+) T cells using only 2 colors, we have defined CD4(+) T cells as MNCs expressing CD4 and not expressing the markers of other MNCs (CD8, CD13, CD14, and CD16) and compared the results to those obtained with the 3-color method in which CD4(+) T cells were defined as CD3(+)CD4(+)CD8(-) MNCs, Both methods produced similar results. An analogous approach was undertaken to enumerate CD8(+) T cells as MNCs expressing CD8 and not expressing the markers of other MNCs. However, when compared with the percents obtained by the 3-color method in which CD8(+) T cells were defined as CD3(+)CD4(-)CD8(+) MNCs, the 2-color method overestimated the percent of CD8(+) T cells. This was likely due to counting CD8(low) null cells as CD8(+) T cells. When the percentages of CD8(high) T cells were evaluated, both methods produced similar results. We conclude that the 2-color method is suitable for the enumeration of CD4(+) T cells and CD8(high) T cells and reserves the third color for further enumeration of CD4(+) or CD8(high) T cell subsets. (C) 1998 Wiley-Liss, Inc.