Validation of blood collection procedures for the determination of circulating vascular endothelial growth factor (VEGF) in different blood compartments

Aims of the study. Studies on circulating VECF have reported mixed results, possibly due to a lack of standardization of the pre-analytical phase. The aim of our investigation was to standardize the sampling procedure for the determination of VECF in different blood fractions. Basic procedures. We evaluated various clotting times for obtaining serum i n 30 subjects, as well as different procedures for the preparation of plasm a Edinburgh anticoagulant mixture (EDTA, PGE1, theophylline) and CTAD. VECF was also assayed in lysed whole blood. In vitro platelet activation was mo nitored by measuring the levels of PF4. VEGF and PF4 were measured using co mmercially available enzyme-linked immunoassays, Main findings. Clotting time increased the release of VEGF, which reached a plateau between 2 and 4 hours. The percent increase of VEGF at 2 hours ran ged from 118% to 4515% (median 327%) compared to samples centrifuged within 10 min from withdrawal. VEGF was not different and PF4 was very low or und etectable in Edinburgh plasma and CTAD plasma, while it was significantly h igher in sodium citrate plasma. VEGF in CTAD plasma was not correlated with platelet count or leukocytes. Serum VECF did not correlate with the leukoc yte number, but it correlated significantly with the platelet count. Principal conclusions. The procedures for sample collection described above are highly standardized and easy to perform in a routine setting. We there fore suggest systematic evaluation of VECF in CTAD plasma, in serum (clotti ng for 2 hours at room temperature) and in whole blood, until prospective c ontrolled clinical studies will have clarified in which blood compartment(s ) VECF provides clinically relevant information.