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 descri bed cytofluorometric methods of apoptosis quantification for phenotypi ng apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma me mbrane integrity can be evaluated following 7-AAD incorporation and th e translocation of phosphatidylserine from the inner to the outer laye r of the plasma membrane can be detected through the FITC-annexin V st aining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed wit h 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 identifie s early apoptotic cells, also characterized as 7-AAD(low) with a reduc ed FSC. Moreover these three methods proved to be reliable and gave st atistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, t he 7-AAD assay easily allowed the identification of debris/apoptotic b odies, which were still stained by anti-cell surface mAbs and might th erefore significantly distort the apoptosis percentage in a given lymp hocyte subset. In the present report we also point out that the ISNT a ssay 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-associat ed decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethano l used for cell permeabilization. Finally, we propose a three step ana lytical strategy to accurately phenotype apoptotic peripheral human ly mphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debr is-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these ob servations emphasize that it is essential to assess critically the abi lity of a cytofluorometric method to phenotype apoptotic cells in comp lex lymphoid populations and that inaccurate identification of cell su bsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure. (C) 1997 Elsevier Science B.V.