A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood

Abstract

Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2-2.5 h, and is expected to detect 0.1-6.0% viable CECs and 0.01-0.20% CPCs within blood mononuclear cell population.

Circulating endothelial progenitor cells and endothelial cells in moyamoya disease

“…However, there are limitations—the relative rarity of these cells and the difficulty in characterizing them simply on the basis of surface antigens. Several authors have used polychromatic flow cytometry which can even better characterize these circulating cells (Duda et al., 2007). Using this protocol, we found that two populations were detectable in numbers (Figure 1).…”

“…The diagnostic criterion for MMD is that cerebral angiography or magnetic resonance angiography must show at least the following findings: (1) stenosis or occlusion of the terminal portion of the intracranial internal carotid artery or proximal portions of the anterior and/or the middle cerebral artery; (2) abnormal moyamoya vessel networks in the vicinity of the occlusive or stenotic lesions in the arterial phase; and (3) bilaterality of findings (1) and (2) must be present (Duda et al., 2007). Sixteen patients presenting with unilateral MMD were included.…”

“…Blood samples were kept at 4°C throughout the procedure. Whole‐peripheral blood samples were analyzed by flow cytometry (Duda et al., 2007). During the procedure, fresh samples were centrifuged at 700  g for 20 min with no brake.…”

“…Lack of uniformity in terminology and methodology has caused considerable confusion in characterizing EPCs, and this may be the predominant reason for the variability in published results. Several authors have used polychromatic flow cytometry to better characterize these circulating cells and have reported that the vast majority of EPCs are comprised of proangiogenic hematopoietic cells that do not display in vivo vessel‐forming ability, but promote recovery of vascular endothelial functions via paracrine mechanisms (Duda, Cohen, Scadden, & Jain, 2007; Khan, Solomon, & McCoy, 2005). …”

VEGF-targeted cancer therapy strategies: current progress, hurdles and future prospects

“…These differences might reflect the prolonged clinical response in rectal cancer patients compared with that of glioblastoma patients, or might be due to disease or agent specificity. Nevertheless, with the development of improved flow cytometry and protein array analysis techniques and subsequent standardization [46,51,61], circulating cell and plasma protein measurements hold promise for identification of valid biomarkers for anti-VEGF therapy.…”