Comparative analysis of apoptosis measured by Hoechst and flow cytometry in non-Hodgkin's lymphomas

Fine-needle samples of 75 non-Hodgkin's lymphomas were investigated for apoptosis immediately and after 24 h of culture after in vitro irradiation (2 Gy, 10 Gy, and nonirradiated controls). Apoptotic cells were simultaneously quantified by fluorescence microscopic enumeration of apoptotic cells using Hoechst 33342 staining, and by flow cytometric detection of sub-G1 peak cells. The nonirradiated controls showed a similar mean percent apoptotic cells using both methods, analyzed immediately (9% by morphology vs. 10% by flow) or after 24 h of culture (40% by morphology vs. 41% by flow). In the irradiated samples, the mean percent apoptotic cells quantified by morphology was higher than by flow cytometry (64% by morphology vs. 55% by flow after 2 Gy irradiation, and 71% vs. 58% after 10 Gy). The results of the two methods were correlated, although large differences were seen between the techniques in individual tumors. In our system, flow cytometric sub-G1 peak analysis appears to underestimate apoptosis. Of these two methods, we find the Hoechst morphology method to be more reliable for quantitation of apoptosis utilizing fresh fine-needle sample material, in that discrimination of apoptotic cells from debris is easier and that both early and late apoptotic cells are detectable.