Quantification of circulating endothelial progenitor cells in human peripheral blood: Establishing a reliable flow cytometry protocol

Objectives: Accurate enumeration of circulating endothelial progenitor cells (CEP) is essential for their potential application as biomarkers of angiogenesis. In this study different stem cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols were assessed for the purpose of CEP quantification. Methods: Blood samples from 19 healthy volunteers and 16 patients with different cancer types were analyzed by means of flow cytometry. Mononuclear cell gating was compared to additional gating on CD45-negative cells. CD34+/KDR+ and CD31+/CD133+ cells were analyzed in a direct immunolabeling approach. CEP were measured at different time points in individual patients and after storage of blood samples for 24 h and 48 h, respectively. Results: In contrast to previous studies, measurement of CD34+/KDR+ cells was an unreliable method for CEP enumeration, regardless of the applied gating strategy. However, detection of CD31+/CD133+ cells in a combined mononuclear cell and CD45-negative gating approach provided a reproducible method for CEP quantification. In individual blood donors, the CEP numbers were stable over time. For the first time, it was demonstrated that CEP are unstable in extracorporeal blood samples. Conclusion: In this study, a reproducible protocol for CEP quantification was established. This protocol should facilitate future studies with the goal to further define the role of CEP as angiogenic biomarkers. (C) 2010 Elsevier B.V. All rights reserved.