Determination of vascular endothelial growth factor (VEGF) in circulating blood: significance of VEGF in various leucocytes and platelets

Aim: The sources of increased vascular endothelial growth factor (VEGF) concentrations in peripheral blood from cancer patients are not known in detail. The aim of the present study was to evaluate correlations between the VEGF content in isolated leucocyte subpopulations and VEGF concentrations in plasma, serum and lysed whole blood. Methods: In 51 colorectal cancer (CRC) patients, circulating T-lymphocytes, B-lymphocytes, monocytes, and granulocytes were isolated by means of immunomagnetic separation. Subsequently, the isolated cells were lysed and VEGF contents in the lysates were determined. In corresponding blood samples, automated complete blood count was performed, and the number of each cell type was correlated to VEGF concentrations in plasma, serum and lysed whole blood. Finally, the impact of increasing clotting time on the release of VEGF to serum was analysed. Results: Isolated neutrophils contained considerable amounts of VEGF. In isolated lymphocytes and monocytes, VEGF was not present in measurable amounts. The number of neutrophils was significantly (p<0.0001) correlated to VEGF concentrations in lysed whole blood, but not to VEGF concentrations in plasma or serum. The number of platelets was significantly correlated to VEGF concentrations in serum and lysed whole blood (p=0.002 and p=0.02, respectively) but not to VEGF concentrations in plasma. Finally, in serum, VEGF concentrations increased with increasing clotting time. However, a plateau was reached between 2 and 6 h of in vitro clotting. Conclusion: Circulating neutrophils contain considerable amounts of VEGF that contribute to high VEGF levels in lysed whole blood. VEGF in circulating platelets contributes to high VEGF levels in serum and lysed whole blood. Allowing whole blood samples to clot for between 2 and 6 h before serum is collected reduces time-dependent, non-uniform release of VEGF.