Storek J, Dawson M A, Maloney D G
Fred Hutchinson Cancer Research Center and University of Washington, Seattle 98104-2092, USA.
Cytometry. 1998 Sep 1;33(1):76-82. doi: 10.1002/(sici)1097-0320(19980901)33:1<76::aid-cyto9>3.0.co;2-9.
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 flow 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 defmed as CD3+CD4-CD8+ MNCs, the 2-color method overestimated the percent of CD8+ T cells. This was likely due to counting CD8low null cells as CD8+ T cells. When the percentages of CD8high 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 CD8high T cells and reserves the third color for further enumeration of CD4+ or CD8high T cell subsets.
通过流式细胞术对血液中CD4 +（单阳性）T细胞进行计数时，容易出现诸如将CD4 +单核细胞或CD4 + CD8 +（双阳性）T细胞误计为CD4 + T细胞等错误。使用三色流式细胞术，将CD4 + T细胞定义为CD3 + CD4 + CD8 - 单核细胞（MNCs）时，可获得相对准确的计数。然而，采用这种方法，进一步将其分类为CD4 + T细胞亚群则需要昂贵的四色流式细胞术。为了仅使用双色来计数CD4 + T细胞，我们将CD4 + T细胞定义为表达CD4且不表达其他MNCs（CD8、CD13、CD14和CD16）标志物的MNCs，并将结果与三色法（将CD4 + T细胞定义为CD3 + CD4 + CD8 - MNCs）所得结果进行比较。两种方法产生了相似的结果。我们采用类似的方法将CD8 + T细胞计数为表达CD8且不表达其他MNCs标志物的MNCs。然而，与三色法（将CD8 + T细胞定义为CD3 + CD4 - CD8 + MNCs）获得的百分比相比，双色法高估了CD8 + T细胞的百分比。这可能是由于将CD8low空细胞计为CD8 + T细胞所致。当评估CD8high T细胞的百分比时，两种方法产生了相似的结果。我们得出结论，双色法适用于CD4 + T细胞和CD8high T细胞的计数，并保留第三种颜色用于进一步计数CD4 +或CD8high T细胞亚群。
