Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in Biobank samples.
BACKGROUND
Blood biomarkers are subject to pre-analytical variability. In many cases, the stability of important new tissue biomarkers during freeze cycles and storage has not been studied sufficiently.


METHODS
To test the stability of matrix metalloproteinases-7 (MMP-7) and their tissue inhibitors (TIMP-1), vascular growth factors (VEGF) and VEGF-receptor, serum samples were frozen and then thawed up to six times. The impact of storage temperature was investigated using an accelerated stability testing protocol. Stability at -20°C and -75°C was calculated using the Arrhenius equation.


RESULTS
The average concentration of TIMP-1 was stable, even after six freeze/thaw cycles. One thawing did not change the concentration of MMP-7 and VEGF-receptor. However, repeated freeze/thaw cycles increased the measured values significantly. Decreases in VEGF concentrations were dramatic, even after the first freeze/thaw cycle. According to the Arrhenius calculation, MMP-7 showed excellent stability, at least 5 years at -20°C and several 100 years at -75°C. The VEGF-receptor maintains 90% of its initial concentration at -20°C over 3 months, and decades at -75°C. TIMP-1 and VEGF showed poor stability with cryopreservation, even at -75°C.


CONCLUSIONS
The stability of MMP-7, TIMP-1, VEGF or VEGF-receptor in biobanking is highly variable, and this should be taken into account in the interpretation of results. A temperature -20°C is unsuitable for prolonged storage of the biomarkers investigated, and repeated thawing of sera is not recommended. VEGF is especially unstable and should be quantitated using serum that has never been frozen.
Immunology Group, Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia. kalle.kisand@ut.ee
Kalle
K
Kisand
Irina
I
Kerna
Jaanika
J
Kumm
Helgi
H
Jonsson
Agu
A
Tamm
D016477:Artifacts; D018070:Biological Specimen Banks; D015415:Biomarkers / Q000097:blood; D015925:Cryopreservation; D004797:Enzyme-Linked Immunosorbent Assay; D005615:Freezing; D006801:Humans; D020783:Matrix Metalloproteinase 7 / Q000097:blood*; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood*; D042461:Vascular Endothelial Growth Factor A / Q000097:blood*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000097:blood*
D015415:Biomarkers; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D020783:Matrix Metalloproteinase 7