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 after surgery. VEGF expression was quantified on immunofluorescence stained slides.


RESULTS
The period of storage and the number of freeze-thaw cycles correlated with an increase in the levels of circulating VEGF (for each anticoagulant but not for serum) and its expression in PBMCs. VEGF expression measured from muscle biopsy sections was higher with freezing delays, with a peak at 30 and 60 min as compared to 15 min.


CONCLUSIONS
The most reliable conditions for measuring both circulating VEGF and its gene expression are to reduce time between blood collection and centrifugation, and to avoid multiple freeze-thaw cycles. Serum collection tubes with no additive and no separator were less sensitive to the pre-analytical variations analyzed in this study. Freezing delay had a significant influence on VEGF protein expression in tissue samples.
1 Unité de Recherche "Génétique Cardiovasculaire", Université de Lorraine , Nancy, France .
Mohsen
M
Azimi-Nezhad
Daniel
D
Lambert
Catherine
C
Ottone
Corinne
C
Perrin
Celine
C
Chapel
Gwenaëlle
G
Gaillard
Michèle
M
Pfister
Christine
C
Masson
Eric
E
Tabone
Fay
F
Betsou
David
D
Meyronet
Marie-Noëlle
MN
Ungeheuer
Sophie Visvikis
SV
Siest
D000925:Anticoagulants / Q000737:chemistry; D019343:Citric Acid / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005615:Freezing; D015870:Gene Expression; D005947:Glucose / Q000031:analogs & derivatives / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods; D013696:Temperature; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D000925:Anticoagulants; D006629:Hirudins; D020033:Protein Isoforms; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C002113:acid citrate dextrose; D019343:Citric Acid; D004492:Edetic Acid; D005947:Glucose