Monochromatic gating method by flow cytometry for high purity monocyte analysis

Authors


  • How to cite this article: Wong L, Davis BH. Monochromatic gating method by flow cytometry for high purity monocyte analysis. Cytometry Part B 2013; 84B: 119–124.

Abstract

Background:

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.

Methods:

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.

Results:

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.

Conclusions:

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

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