title: An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood.
abstract: Circulating adult CD34(+)VEGFR2(+) endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14(+) monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2(+) monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2(+)CD14(+)CD16(mid) angiogenic monocytes and CD34(+)VEGFR2(+)CD45(low/-) EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, and APC-Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE-Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence-minus-one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean +/- SD) of 1.45 +/- 1.29% for Tie2(+)CD14(+)CD16(mid) monocytes (n = 11, range: 0.12-3.64%) and 0.019 +/- 0.013% for CD34(+)VEGFR2(+)CD45(low/-) EPCs (n = 17, range: 0.003-0.042%). The intra- and inter-assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use.
authors.affiliation: Institute for Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, 52074 Aachen, Germany. mhristov@ukaachen.de
authors.forename: Mihail
authors.initials: M
authors.lastname: Hristov
authors.forename: Susanne
authors.initials: S
authors.lastname: Schmitz
authors.forename: Christoph
authors.initials: C
authors.lastname: Schuhmann
authors.forename: Thorsten
authors.initials: T
authors.lastname: Leyendecker
authors.forename: Philipp
authors.initials: P
authors.lastname: von Hundelshausen
authors.forename: Florian
authors.initials: F
authors.lastname: Krötz
authors.forename: Hae-Young
authors.initials: HY
authors.lastname: Sohn
authors.forename: Frans A
authors.initials: FA
authors.lastname: Nauwelaers
authors.forename: Christian
authors.initials: C
authors.lastname: Weber
mesh_terms: D000368:Aged; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008297:Male; D009000:Monocytes / Q000166:cytology*; D018919:Neovascularization, Physiologic; D042787:Receptor, TIE-2 / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D018950:Lipopolysaccharide Receptors; D017452:Receptors, IgG; D042787:Receptor, TIE-2; D040301:Vascular Endothelial Growth Factor Receptor-2; D017493:Leukocyte Common Antigens