Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods.

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD(low) with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.