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.