Apoptosis of cells in aged samples as detected by the ProCOUNT reagent

An unusual population of high side scatter, low nucleic acid dye binding, dim CD45 cells was found in aged blood samples stained with the ProCOUNT reagent. Cell surface staining showed that these cells have the surface phenotype of neutrophils. However, they have decreased expression of several surface antigens, bind annexin V, and stain more dimly than normal neutrophils with LDS-751. These characteristics indicate that the cells have become apoptotic. The decreased expression of the CD45 antigen on apoptotic neutrophils could have an impact on some methods for enumerating CD34-positive progenitor cells. Absolute CD34-positive cell counts are frequently obtained by multiplying CD34-positive cells as a percentage of the total cells by the white blood cell count from a hematologyanalyzer. Cells staining dimly with CD45 may not pass a flow cytometer threshold set on this parameter but appear to be detected by a hematology instrument thresholding on cell size. Thus the white blood cell counts from the two sources may not be identical, introducing error into the calculated absolute CD34 count. Absolute counts of CD34 cells based on simultaneous acquisition of a counting bead are not affected by the presence of this population, but purity estimates can be affected if the possible presence of these cells is not considered in aged samples.