optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood

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⁺CD16mid angiogenic monocytes and CD34⁺VEGFR2⁺CD45low/⁻ 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⁺CD16mid monocytes (n = 11, range: 0.12-3.64%) and 0.019 ± 0.013% for CD34⁺VEGFR2⁺CD45low/⁻ 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. © 2009 International Society for Advancement of Cytometry