Fluorescence activated cell sorting for simultaneous assessment of nine surface markers of circulating endothelial progenitor cells in pulmonary hypertension

Background: The role of circulating endothelial progenitor cells (EPCs) in pulmonary hypertension (PH) patients is unknown. In this pilot study we established a nine-colour staining assay for the Fluorescent Activated Cell Sorting (FACS) to characterize the circulating EPCs in PH patients as compared to healthy controls. Patients and methods: Peripheral and central venous blood was taken from PH patients and healthy controls. Mononuclear blood cells were isolated by means of density gradient centrifugation. The cells were simultaneously stained with fluorescent conjugated antibodies against the cell surface markers c-kit, CXCR2, VEGFR2, CD34, CD14, CD31, CD133, CD16, and CD45. EPCs were defined as CD34+ CD133+ VEGFR2+ cells. Results: N=10 PH patients (n=4 idiopathic, n=3 chronic thromboembolic, n=3 left heart disease), mean pulmonary artery pressure: 42 ± 14 mmHg, pulmonary vascular resistance: 539 ± 290 dyn·s/cm5) and n=10 healthy controls were included. No spectral overlap occurred during the nine-colour staining assessment. Circulating EPCs counted from peripheral and central blood revealed no significant differences. All cells were CD45+, suggesting their hematopoietic origin. CD34+ cells were significantly decreased in PH patients as compared to controls (0.3 % vs. 1.8 % of the mononuclear gate, p<0.005). EPCs were significantly lower in PH patients vs. control (2% vs. 44% of CD34+CD133+ cells, p<0.0001). Conclusion: These preliminary results suggest that multi-colour FACS is suitable for EPC quantification and characterization. Further studies are necessary to define distinct circulating EPCs as markers of PH.