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.