An improved assay for enumeration of circulating endothelial cells

Circulating endothelial cells have been established as markers of vascular disease, such as small vessel vasculitis, acute vascular rejection in renal transplant recipients, and cyclosporine-induced endothelial damage. Enumeration of these cells by immunomagnetic isolation and acridine staining remains the gold standard but necessitates considerable experience and expenditure. A simpler test would therefore be of great utility. Hence, our aim was to develop an improved simple assay to enumerate endothelial cells in peripheral blood. We had already used various surface markers to corroborate the endothelial origin of cells. Here, we studied the enumeration of cell numbers with immunomagnetic isolation and a variety of subsequent stains, such as CD31, von Willebrand's factor (vWF) immunocytochemistry, and Ulex europaeus lectin-1 (UEA-1). Eventually, we devised a simple protocol for enumeration using immunomagnetic isolation and a subsequent UEA-1 lectin stain. We evaluated the use of this protocol in parallel to immunomagnetic isolation and acridine counting alone in 92 renal transplant recipients who underwent renal biopsy. Recovery of various concentrations of human umbilical vein endothelial cells from blood was also studied. Immunomagnetic isolation and subsequent UEA-1 staining permits easier enumeration of circulating endothelial cells in peripheral blood. The assay is simple and easy to use, thus allowing for a more widespread use of circulating endothelial cells as a marker of vascular damage.