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

Abstract

Circulating adult CD341 VEGFR2 1 endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD141 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 1 monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2 1 CD14 1 CD16 mid angiogenic monocytes and CD34 1 VEGFR2 1 CD45 low/2 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 AE SD) of 1.45 AE 1.29% for Tie2 1 CD14 1 CD16 mid monocytes (n ¼ 11, range: 0.12-3.64%) and 0.019 AE 0.013% for CD34low/2 EPCs (n ¼ 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

Large platelets but not putative endothelial progenitor cells are associated with low strut coverage after drug-eluting stent implantation

“…In the study of Hristov et al, dead cells were excluded together with high CD45 expression in order to quantify CD34 and KDR expression [30]. Although their data is not directly comparable due to different gating strategies, a very low number of CD34 + KDR + CD45 −/dim cells was observed in congruence with our data.…”

Endothelial progenitor cells in patients with chronic obstructive pulmonary disease

“…The EC-CFU assay was originally employed to overcome the difficulties associated with accurately quantifying small numbers of circulating cells by flow cytometry. However, while providing a measure of vascular health, it is now known that these colonies do not arise directly from circulating EPC and in fact are the progeny of myeloid cells that indirectly contribute to tissue angiogenesis via paracrine mechanisms (4,8). EC-CFU are known to express a variety of endothelial characteristics, but are predominantly composed of activated leukocytes of myeloid and lymphocytic lineage.…”

Stepwise Optimization of the Procedure for Assessment of Circulating Progenitor Cells in Patients with Myocardial Infarction

“…In conclusion, although the interest in CPCs is steadily increasing, the lack of a consensus on standardised protocols remains a barrier to clinical exploitation [21], [22]. In this study, we addressed some critical steps instrumental to optimal enumeration and characterisation of CPCs in cardiovascular patients.…”