COMPARATIVE-ANALYSIS OF APOPTOSIS MEASURED BY HOECHST AND FLOW-CYTOMETRY IN NON-HODGKINS-LYMPHOMAS

Fine-needle samples of 75 non-Hodgkin's lymphomas R-ere investigated f or apoptosis immediately and after 24 h of culture after in vitro irra diation (2 Gy, 10 Gy, and nonirradiated controls). Apoptotic cells wer e simultaneously quantified by fluorescence microscopic enumeration of apoptotic cells using Hoechst 33342 staining, and by flow cytometric detection of sub-G(1) peak cells, The nonirradiated controls showed a similar mean percent apoptotic cells using both methods, analyzed imme diately (9% by morphology vs. 10% by flow) or after 24 h of culture (4 0% by morphology vs. 41% by flow). In the irradiated samples, the mean percent apoptotic cells quantified by morphology was higher than by f low cytometry (64% by morphology Fs. 55% by flow after 2 Gy irradiatio n, and 71% vs. 58% after 10 Gy), The results of the two methods were c orrelated, although large differences were seen between the techniques in individual tumors, In our system, flow cytometric sub-G(1) peak an alysis appears to underestimate apoptosis, Of these two methods, we fi nd the Hoechst morphology method to be more reliable for quantitation of apoptosis utilising fresh fine-needle sample material, in that disc rimination of apoptotic cells from debris is easier and that both earl y and late apoptotic cells are detectable, (C) 1998 Wiley-Liss, Inc.