A new approach for rapid and reliable enumeration of circulating endothelial cells in patients

Background:  Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300per mL). Objectives:  Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment. Methods:  CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4mL needed for a precise enumeration of CECs at a frequency of <1cellμL -1. Results:  The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch ®). CEC levels ranged from 4 to 79CECmL -1 in healthy individuals and were significantly higher in patients with advanced solid malignancies (P=0.0008) and in patients with hematological malignancies (P<0.0001). Conclusions:  This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.

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