Assays of antigen expression on myeloid cells have an underlying premise that the assay integrates high purity gating of the leukocyte subpopulation in question. While CD45/side scatter (SSC) gating provides sufficient gating purity for qualitative assays of antigen expression; it is unsuitable for quantitative assays of antigen changes, especially monocytes. We have validated a monochromatic gating approach combining CD45 and CD64 labeled with the same fluorochrome that allows for high purity monocyte gating.
Twenty-five blood samples were stained using three different antibody combinations (CD45 FITC + CD163 PE; CD45 FITC + CD64 PE; CD45 FITC + CD64 FITC). Data analysis focused on the percentage of “monocytes” defined by the various antibody and SSC gating combinations.
Percent monocyte recovered by monochromatic CD64 gating was not statistically different from two-color CD45 + CD64 or CD45 + CD163 gating. All three methods of immunologic monocyte identification yielded a 12.93%–15.15% reduction in the “monocyte” percentage compared to CD45/SSC gating.
A monochromatic combination of CD45 and CD64 antibodies with scatter signals allows higher purity monocyte gating by flow cytometry (FC) compared to CD45/SSC gating. This approach allows for the development of a high resolution four-color assay, such as for detection of paroxysmal nocturnal hemoglobinuria, whereby a single four-color tube will allow simultaneous high purity monocyte (CD64+) and neutrophil (CD15+) analysis of both phosphatidylinositol (PI) linked protein expression and FLAER binding. © 2013 International Clinical Cytometry Society