Quantification of apoptosis events in myeloid leukemia cells. Leukemia cells from pediatric AML patients were analyzed before and after culture with or without the caspase inhibitor zVADfmk by flow cytometry. (A) Identification of leukemia cells and analysis of cell death parameters. Leukemia cells were identified by surface markers (i: CD33 or CD34, gate LC) and further analysis was conducted gated on these cells. Cell death (cd) was estimated by forward/side scatter profile principally detecting apoptotic cells by changes of decreased volume and increased light scattering (ii). Activated caspase-3 (ac) and cytochrome c release (cc) were analyzed by simultaneous intracellular staining (phycoerythrin (PE)-conjugated anti–active caspase-3 and anti–cytochrome c 7H8.2C12 antibody, the latter followed by fluorescein-isothiocyanate (FITC)-conjugated goat anti–mouse IgG2b antibody) and quantified in the ac versus cc plot (iii). The staining was established using isotype-matched fluorochrome-conjugated unspecific antibodies. Cells with active caspase-3 were quantified as percentage of events in the upper and lower right quadrants (ac), cytochrome c release was estimated counting events in the lower left and right quadrants (cc). Because caspase-3 activation is completely caspase dependent, only the zVAD inhibitable caspase activation was considered (ac: difference of ac values after culture with and without zVAD); caspase dependent cytochrome c release (cc^dep: difference of cc values after culture with and without zVAD) was discriminated from independent (cc^indep: difference of cc values before and after culture with zVAD) and total cytochrome c release (cc^total, sum of cc^dep and cc^indep). (B) Distinct patterns of apoptosis signaling. Cytochrome c versus active caspase-3 plots for 2 patient samples showing cytochrome c release dependent (i) or independent (ii) on upstream or amplifier caspases such as caspase-8, resulting in positive (i) or negative (ii) CRAC^dep values. (C) Treatment outcome in different patient groups according to CRAC^dep. Superior relapse-free survival (i) and superior continuing remission (ii) of CRAC^dep-positive pediatric AML patients. Kaplan Meier analysis, P indicates significance.