Measurement of Human Endothelial Cells in Whole Blood

To evaluate the usefulness of measuring circulating endothelial cells as an in vivo indicator for vascular injury, studies were performed in vitro using cultured human endothelial cells and Factor VIII-related antigen (VIIIR:Ag) as the specific cell marker. Cultured human endothelial cells mixed with normal whole blood were processed by Ficoll-Hypaque gradient sedimentation to remove red cells and granulocytes, followed by pellet formation of the mononuclear supernatant fraction to separate the bulk of platelets. Cell films, prepared from a measured volume of the final cell suspension, were stained with fluorescent antibody against VIII R:Ag, and positively stained cells were counted under fluorescence microscopy. The final recovery of endothelial cells was 64.8 +/- 19.3% (n = 41), and the minimal detectable concentration of endothelial cells was about 0.02 cells/microliter whole blood. No endothelial cells were detected in the blood of normal individuals.