A SENSITIVE METHOD FOR QUANTIFYING CYTOMEGALIC ENDOTHELIAL-CELLS IN PERIPHERAL-BLOOD FROM CYTOMEGALOVIRUS-INFECTED PATIENTS

A sensitive method has been developed for the quantification of cytome galic endothelial cells (CEC) in peripheral blood (PB) of patients wit h active cytomegalovirus (CMV) infections. The three subsequent key st eps of this method are density centrifugation to enrich endothelial ce lls (EC) in the mononuclear cell (MNC) fraction, EC-specific staining, and fluorescence-activated cell sorting (FACS) of EC onto adhesion sl ides. The FAGS method was compared with the conventional method of cyt ocentrifugation of the MNC fraction onto slides, followed by EC-specif ic staining. The main advantage of the additional steps for the isolat ion and quantification of CEC in PB by FAGS is a 10-times-greater sens itivity than by cytocentrifugation of the MNC fraction alone. The reco very percentages of EC from whole blood were comparable for both metho ds. Recoveries of EC obtained after FAGS were 53% +/- 16.5%, (mean +/- standard deviation), and recoveries of EC obtained after cytocentrifu gation of the MNC fraction were 43% +/- 4.3%, In patients with active CMV infection, 5 to 72 CEC were detected by FAGS, equivalent to 0.8 to 9.0 CEC/ml of blood. With this method for isolation and quantificatio n, the characterization of CEC in PB of patients with CMV-associated c linical symptoms, as well as the quantification of EC in PB of patient s with pathophysiological manifestations involving endothelial damage that are different from those caused by CMV infections, can be perform ed.