Validation of blood collection procedures for the determination of circulating vascular endothelial growth factor (VEGF) in different blood compartments.
OBJECTIVE
Studies on circulating VEGF 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 VEGF in different blood fractions.


METHODS
We evaluated various clotting times for obtaining serum in 30 subjects, as well as different procedures for the preparation of plasma Edinburgh anticoagulant mixture (EDTA, PGE1, theophylline) and CTAD. VEGF was also assayed in lysed whole blood. In vitro platelet activation was monitored by measuring the levels of PF4. VEGF and PF4 were measured using commercially available enzyme-linked immunoassays.


RESULTS
Clotting time increased the release of VEGF, which reached a plateau between 2 and 4 hours. The percent increase of VEGF at 2 hours ranged from 118% to 4,515% (median 327%) compared to samples centrifuged within 10 min from withdrawal. VEGF was not different and PF4 was very low or undetectable in Edinburgh plasma and CTAD plasma, while it was significantly higher in sodium citrate plasma. VEGF in CTAD plasma was not correlated with platelet count or leukocytes. Serum VEGF did not correlate with the leukocyte number, but it correlated significantly with the platelet count.


CONCLUSIONS
The procedures for sample collection described above are highly standardized and easy to perform in a routine setting. We therefore suggest systematic evaluation of VEGF in CTAD plasma, in serum (clotting for 2 hours at room temperature) and in whole blood, until prospective controlled clinical studies will have clarified in which blood compartment(s) VEGF provides clinically relevant information.
Dittadi
R
R
Center for the Study of Biological Markers of Malignancy, Regional General Hospital ULSS 12, Venice, Italy.
Meo
S
S
Fabris
F
F
Gasparini
G
G
Contri
D
D
Medici
M
M
Gion
M
M
D014408:Biomarkers, Tumor / Q000097:blood*; D001800:Blood Specimen Collection / Q000379:methods; D016228:Endothelial Growth Factors / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D007958:Leukocyte Count; D008222:Lymphokines / Q000097:blood*; D009369:Neoplasms / Q000097:blood*; D009360:Neoplastic Cells, Circulating; D009389:Neovascularization, Pathologic / Q000097:blood; D010976:Platelet Count; D010978:Platelet Factor 4 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D014408:Biomarkers, Tumor; D016228:Endothelial Growth Factors; D006454:Hemoglobins; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D010978:Platelet Factor 4