Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation

Background: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. Methods: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). Results: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. Conclusions: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis. (c) 2006 International Society for Analytical Cytology.