Effect of Repeated Freezing and Thawing on Biomarker Stability in Plasma and Serum Samples

OBJECTIVES: The stability of circulating proteins can be affected by repeated freezing and thawing. The aim of our study was to identify the effect of repeated freezing and thawing on the plasma and serum concentrations of eight proteins [interferon-γ, interleukin (IL)-8, IL-15, IL-17A, matrix metalloproteinase (MMP)-7, tumor necrosis factor-α, vascular endothelial growth factor (VEGF), and VEGF receptor 2 (VEGF-R2)]. METHODS: We assessed the concentration changes of these proteins in 30 plasma and serum samples subjected to three, four, or five freeze–thaw cycles, and compared these with the concentration changes in the samples that were subjected to two freeze–thaw cycles before analysis. RESULTS: Repeated freezing and thawing by up to five cycles did not modify the plasma and serum concentrations of interferon-γ, IL-8, and VEGF-R2, while levels of MMP-7, tumor necrosis factor-α, and VEGF were significantly changed in both plasma and serum samples. Moreover, MMP-7 and VEGF concentrations tended to increase with freeze–thaw cycles. They were more elevated in plasma samples (up to about 15%) than in serum samples (up to about 7%), suggesting that serum is the preferred sample type for the analysis of circulating proteins. CONCLUSION: This is the first report on the effect of repeated freezing and thawing on plasma concentrations of MMP-7 and VEGF-R2. Our findings propose that researchers should consider the number of freeze–thaw cycles to select plasma or serum samples, depending on the type of analyte.