Effect of Repeated Freezing and Thawing on Biomarker Stability in Plasma and Serum Samples.
Author(s): Lee JE, Kim SY, Shin SY
Publication: Osong Public Health Res Perspect, 2015, Vol. 6, Page 357-62
PubMed ID: 26835245 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of additional freeze-thaw cycles on levels of eight biomarkers in serum and plasma freeze-thaw cycled twice.
Conclusion of Paper
Plasma and serum levels of matrix metalloproteinase (MMP)- 7 and vascular endothelial growth factor (VEGF) tended to increase with each additional freeze-thaw cycle and, with the exception of VEGF in serum, were significantly higher after three or more cycles than after two cycles. While plasma levels of tumor necrosis factor-α (TNF-α) levels declined slightly with three or more freeze-thaw cycles, levels of TNF-α were higher in serum freeze-thaw cycled three or four times than that freeze-thawed twice. Interleukin (IL)-15 and IL-17A levels were unaffected by additional freeze-thaw cycling of plasma but were higher in serum specimens subjected to three freeze thaw cycles than that freeze-thaw cycled twice and levels of IL-15 were also higher in serum freeze-thaw cycled four times than that freeze-thaw cycled twice. Plasma and serum levels of interferon-gamma (IFN-γ), IL-8, and VEGF-receptor 2 (VEGF-R2) were unaffected by additional freeze-thaw cycling.
Studies
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Study Purpose
This study compared the effects of additional freeze-thaw cycles on levels of IFN-γ, three interleukins, MMP-7, TNF-α, VEGF, and VEGF-R2 in serum and plasma freeze-thaw cycled twice. Leftover blood specimens (diagnosis unspecified) collected in K2EDTA plasma separator tubes and vacutainer serum separator tubes were centrifuged at 2000 x g for 15 min to produce plasma and serum, respectively. To achieve sufficient specimen volume, 2-3 specimens were mixed together to produce 30 pools and then stored at -75˚C. Specimens were thawed at 37˚C, aliquoted into four tubes, and then refrozen at -75˚C. Specimens were thawed one, two, three, or four additional times before analysis. Cytokine and VEGF levels were quantified using a magnetic bead panel immunoassay. MMP2 and VEGF-R2 levels were determined by ELISA.
Summary of Findings:
Plasma and serum levels of MMP-7 and VEGF tended to increase with each additional freeze-thaw cycle and, with the exception of VEGF in serum, were significantly higher after three or more cycles than after two cycles (P<0.05). Plasma and serum freeze-thaw cycled five times had higher levels of MMP-7 (15.6% and 6.5%, respectively) and VEGF (15.1% and 7.0%) than that cycled two times. Plasma TNF-α levels were significantly lower in specimens freeze-thawed three or more times than those freeze-thawed twice, but had only declined by 3.2% after 5 cycles from levels measured after 2 cycles. In contrast, levels of TNF- α were higher in serum freeze-thaw cycled three or four times, but not five times, than that freeze-thawed twice (3.4%, P<0.05 after three cycles and 4.9%, P<0.05 after 4 cycles). IL-15 and IL-17A levels were unaffected by additional freeze-thaw cycling of plasma and were comparable in serum freeze-thawed twice versus five times; however, serum specimens had significantly higher levels of IL-15 after three and four freeze-thaw cycles than those freeze-thaw cycled twice (8.7% P<0.05 and 12.4%, P<0.05, respectively) and IL-17a were also higher after three freeze-thaw cycles compared to specimens freeze-thawed twice (17.2% P<0.05). Plasma and serum levels of IFN- γ, IL-8, and VEGF-R2 were unaffected by additional freeze-thaw cycling.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein ELISA Protein Immunoassay Glycoprotein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 2 cycles
3 cycles
4 cycles
5 cycles
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum