Optimized flow cytometric analysis of endothelial progenitor cells in peripheral blood.

Although flow cytometry is a rapid and convenient way to measure the number of circulating endothelial progenitor cells (EPC), there is no standard technique for preparation and measurement. The aim of this study was to present an optimized preparation method for EPC measurement which should serve as a standard to facilitate the comparison of the results in stem cell investigations by different research groups. We have looked for the preparation method which delivered the best immunostaining with the directly conjugated antibodies against VEGF R2, CD133, CD34, and CD45. In order to test the sensitivity of the method, we determined the number of EPC in the peripheral blood of volunteers by flow cytometry and by cell culture assay. Furthermore, we have evaluated the influence of different durations of conservation on the EPC cell count. The pre-treatment of blood samples with 0.2% formaldehyde for 30 minutes delivers the best immunostaining, and blood samples can be stored overnight at 4 degrees C without loss of counting rate for EPC. We found an excellent correlation (r = 0.98) between the flow cytometric measurement and the cell count of the cell culture method. The presented protocol for the flow cytometric measurement of EPC in the peripheral blood can be used as a diagnostic or prognostic tool; we propose this protocol as the standard for EPC quantification.