Monochromatic gating method by flow cytometry for high purity monocyte analysis
- 2 January 2013
- journal article
- Published by Wiley in Cytometry Part B: Clinical Cytometry
- Vol. 84B (2), 119-124
- https://doi.org/10.1002/cyto.b.21053
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 SocietyKeywords
This publication has 14 references indexed in Scilit:
- Flow cytometric testing for paroxysmal nocturnal hemoglobinuria: CD64 is better for gating monocytes than CD33Cytometry Part B: Clinical Cytometry, 2012
- Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometryCytometry Part B: Clinical Cytometry, 2012
- Comparative assessment of the recognition of domain-specific CD163 monoclonal antibodies in human monocytes explains wide discrepancy in reported levels of cellular surface CD163 expressionImmunobiology, 2011
- Nomenclature of monocytes and dendritic cells in bloodBlood, 2010
- Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometryCytometry Part B: Clinical Cytometry, 2010
- Comparative Study of Monocyte Enumeration by Flow Cytometry: Improved Detection by Combining Monocyte-Related Antibodies with Anti-CD163Laboratory Hematology, 2004
- Blood Monocytes Consist of Two Principal Subsets with Distinct Migratory PropertiesImmunity, 2003
- Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometryCytometry, 1996
- Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14.The Journal of Experimental Medicine, 1991