Comparative measurement of spontaneous apoptosis in pediatric acute leukemia by different techniques

Background To distinguish between subgroups of patients with acute leukemia, the rate of spontaneous (culture‐induced) apoptosis of leukemic cells was evaluated using five methods. Methods Leukemic cells (cells) from the bone marrow of children with acute lymphoblastic leukemia (ALL, n = 112) and acute myeloid leukemia (AML, n = 30) were cultured for 20 h in vitro. The level of apoptosis was detected by fluorescent microscopy after staining with acridine orange (AO) or by flow cytometry after staining using PI, JC‐1, the APO‐BRDU kit, or the AnnexinV‐FITC kit. Results ALL cells were significantly more sensitive to spontaneous apoptosis versus AML cells, as was detected by all methods. The least sensitive technique was apoptosis detection by sub‐G1‐peak/PI‐staining. No difference in the rate of apoptosis in cells was determined between T‐ and B‐lineage ALL patients. In patients with B‐lineage ALL, strong positive correlation existed between the level of cells with loss of mitochondrial membrane potential (JC‐1), chromatin condensation (AO), and externalization of phosphatidylserine (AnnexinV+PI+). The proportion of AnnexinV+PI– cells had no correlative link with any other apoptotic cell subpopulation. Conclusions We found different sensitivities of ALL and AML cells to undergoing spontaneous apoptosis in vitro. Detection of the early/intermediate, but not the late stage of apoptosis is of preferable for correct assignment of spontaneous apoptosis in pediatric acute leukemia. Cytometry Part B (Clin. Cytometry) 56B:16–22, 2003. © 2003 Wiley‐Liss, Inc.