Influence of Pre-analytical Variables on VEGF Gene Expression and Circulating Protein Concentrations

Background: The extended role of vascular endothelial growth factor (VEGF) in human pathophysiology led us to evaluate pre-analytical parameters possibly influencing its levels in peripheral blood and tissues. The effects on VEGF protein levels and mRNA expression were measured after storage delay (blood and tissue), use of different types of anticoagulants (blood), and after different numbers of freeze-thaw cycles (blood). Methods: Blood from healthy donors was sampled simultaneously in ethylene diamine tetraacetic acid (EDTA), acid citrate dextrose (ACD-A), hirudin, and serum separation tubes. For each anticoagulant, VEGF was measured by enzyme-linked immunosorbent assay (ELISA) with different conditions of delay at 4°C before centrifugation (2 h, 4 h, or 48 h) and of different numbers of freeze-thaw cycles (1, 2, and 10). The transcripts coding for the VEGF165 isoform were quantified in peripheral blood mononuclear cells by RT-PCR. Muscle biopsy samples were frozen with delays of 15, 30, or 60 min afte...