Cells meeting our immunophenotypic criteria of endothelial cells are large platelets

Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)(low-to-intermedate), sideward light scatter (SSC)(low), CD45(-), CD31(++) and CD146(+) is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658-3661). Here, we set out to confirm the endothelial nature of these cells.

We isolated cells with a FSC(low-to-intermediate), SSC(low), CD31(++), CD45(dim) immunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel-Palade bodies in the sorted cells.

CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel-Palade bodies, and revealed CMOIC to be large platelets.

The vast majority of cells with the immunophenotype FSC(low-to-intermediate), SSC(low), CD45(-), CD31(++) do not express CD146 and are large platelets rather than endothelial cells.