COMPARATIVE-ANALYSIS OF FLOW CYTOMETRIC METHODS FOR APOPTOSIS QUANTITATION IN MURINE THYMOCYTES AND HUMAN PERIPHERAL LYMPHOCYTES FROM CONTROLS AND HPV-INFECTED PERSONS - EVIDENCE FOR INTERFERENCE BY GRANULOCYTES AND ERYTHROCYTES

The present article reports a multiparametric cytofluorimetric analysi s of apoptosis in murine thymocytes and human PBMC from healthy donors or HN-infected patients. We have evaluated four previously described cytofluorimetric methods of apoptosis quantification, each of them det ecting distinct cellular alterations of the apoptosis process. Reduced DNA stainability was detected with the PI assay on nuclei and the AO/ EB dual staining method was evaluated on entire and non-fixed cells. D NA strand breaks were detected following in situ nick translation, and alterations in membrane integrity were evaluated following 7-AAD inco rporation. When apoptosis was quantified in murine thymocytes under va rious conditions of induction, the combined analysis of FSC/SSC criter ia and 7-AAD or AO/EB staining on the same samples permitted the ident ification of distinct steps in the apoptosis process. Moreover these f our methods proved to be reliable and gave statistically similar resul ts both on murine thymocytes and PBMC from healthy donors. However, in HIV-infected persons, some discordant apoptosis determinations were o bserved with PI and 7-AAD staining assays. We found that after Ficoll isolation, PBMC from AIDS patients were enriched in erythrocytes and g ranulocytes. On the one hand granulocytes were found to be responsible for a poor apoptosis estimation with the PI assay whereas erythrocyte s were responsible for an underestimation rate of apoptosis in the 7-A AD assay. To prevent such interference, we propose some modifications which render these methods more suitable for application to PBMC from HIV-infected patients. Taken together these observations indicate that it is essential to assess critically the apoptosis quantification met hods with respect to their applicability to complex lymphoid populatio ns such as those from AIDS patients.