Flow cytometric evaluation of circulating endothelial cells: A new protocol for identifying endothelial cells at several stages of differentiation

Several factors may influence the analysis of endothelial cells (ECs) by flow cytometry: separation of mononuclear cell, washing and centrifugation steps, panel of monoclonal antibodies, and the lack of standardization of gating technique. Therefore, the reliable quantification of ECs remains a technical challenge. The purpose of this study is to define a new flow cytometric protocol to characterize and quantitate ECs. In previous investigations, increased numbers of circulating ECs have been found in sickle cell disease. The patients with sickle cell disease might provide useful material for the study. We performed flow cytometry on whole blood from 20 normal controls and 31 patients with sickle cell disease (20 patients with steady-state disease and 11 patients with vaso-occlusive crises) using a lyse/no-wash procedure, specific and nonspecific antibody combinations (CD146, CD144, CD34, and CD117), and broad gating. This protocol produced much higher values for the number of circulating ECs (a mean of 2,396.55 +/- 658.37 ECs/mL in controls vs 6,709.60 +/- 1,772.32 ECs/mL in the steady-state group, or 18,213.50 +/- 8,451 in the vaso-occlusive crises group, P < 0.001 for both), and also showed variable EC size and granularity, which may reflect activated, or early release ECs. This novel protocol performed comparably in terms of reproducibility, reliability, and dilution linearity with a previously described protocol. This approach has significant advantages for the characterization and quantitation ECs compatible with the pathophysiology. Using the specific antibodies, CD146 and CD144, together may give more informative EC data than the general approach used.