Pro-apoptotic and anti-apoptotic Bcl-2–related protein expression in MDS/MDS-AML and normal controls.

BM samples were incubated with FITC-conjugated anti-CD34 Mab and fixed with formaldehyde. To prevent nonspecific binding of Mabs to Fc receptors, cells were pre-incubated with rabbit-anti-mouse (RAM) immunoglobulins, permeabilized, and then incubated with mouse-anti-human Mabs to Bax, Bad, Bcl-2, and Bcl-X on ice. Washed cells were subsequently incubated with PE-conjugated RAM on ice prior to flow cytometric analysis. The mean fluorescence intensity (MFI) value for each Bcl-2–related protein was calculated by dividing the MFI value of the positively labeled cells by that of cells stained with an isotype control antibody. To determine individual Bcl-2–related protein levels, we calculated an Oncoprotein Index (OPI) whereby OPI equals the product of the percentage of positive cells and MFI. Median Bax, Bad, Bcl-2, and Bcl-X OPI are plotted in specific diagnostic subgroups.