In this study, we assessed whether the plasma and serum concentrations of eight different proteins are affected by repeated freezing and thawing. The levels of IFN-γ, IL-8, and VEGF-R2 were stable in both plasma and serum samples during repeated freeze–thaw cycles in our experimental conditions. There are a few reports on the effect of repeated freeze–thaw cycles on plasma and serum concentrations of IFN-γ, IL-8, and VEGF-R2
9,
12,
13,
14, except for the plasma concentration of VEGF-R2. Our findings are concordant with these publications, showing that these proteins are not susceptible to degradation induced by repeated freeze–thaw cycles. In addition, we determined for the first time that VEGF-R2 is also stable in plasma samples during repeated freeze–thaw cycles. IFN-γ, IL-8, and VEGF-R2 have attracted attention as new biomarkers of various diseases such as ovarian carcinoma, urinary bladder cancer, acute pyelonephritis, osteoarthritis, or rheumatoid arthritis
15,
16,
17,
18,
19,
20. Our study shows that these may become stable biomarkers, which can be used for diagnosis or prediction of prognosis, regardless of repeated freezing and thawing of samples.
MMP-7, TNF-α, and VEGF levels were significantly changed in both plasma and serum samples during repeated freeze–thaw cycles. MMP7 and VEGF levels were elevated by approximately 15% in plasma samples and by 7% in serum samples after five freeze–thaw cycles, compared with two freeze–thaw cycles. MMP7 and VEGF levels showed a tendency to increase with freeze–thaw cycles. In a previous study, changes in the MMP-7 level induced by repeated freezing and thawing were determined in serum samples
[9], concordant with our results. We identified for the first time that the MMP7 level was elevated by repeated freezing and thawing in serum as well as in plasma samples. MMP7 has attracted attention as a new biomarker for cancer, joint diseases, and liver diseases
21,
22,
23. Concentrations of MMP7 are increased in the serum of rheumatoid arthritis patients with interstitial lung disease
[22] and in the plasma of patients with asymptomatic interstitial lung disease
[21]. Therefore, we recommend that researchers should consider the number of freeze–thaw cycles to select plasma or serum samples for MMP7 analysis. Azimi-Nezhad et al
[24] reported that plasma VEGF levels changed as a result of repeated freezing and thawing. Guo et al
[13] identified that concentrations of VEGF did not change in the plasma samples for up to 10 freeze–thaw cycles, compared with unfrozen samples, which was in contrast to our results. Guo et al
[13] collected whole blood into lithium heparin tubes, unlike our and other studies, which use ethylenediaminetetraacetic acid tubes. Taken together, these facts indicate that the effect of repeated freezing and thawing on the stability of plasma VEGF may be different depending on the tube type used for sample collection. In the case of serum, it has been reported that VEGF levels are not affected by repeated freezing and thawing
13,
24; however, our study shows that VEGF levels were slightly elevated after five freeze–thaw cycles. We thawed serum samples at 37°C, but in other studies, samples were thawed at room temperature, implying that the thawing temperature of serum samples may influence the stability of circulating proteins. Thus, sample thawing should occur at temperatures as low as possible. In addition, we determined that serum concentrations of MMP-7 and VEGF are less affected than that in plasma samples, suggesting that serum is the preferred sample for the analysis of circulating proteins.
In conclusion, our study shows the different effects of repeated freezing and thawing on the stability of eight circulating proteins. IFN-γ, IL-8, and VEGF-R2 are not susceptible to freeze–thawing-induced protein concentration changes, while MMP7, TNF-α, and VEGF are slightly susceptible. Furthermore, we identified that the tube type used for collection of whole blood and the thawing temperature of samples may influence the stability of the circulating proteins. We believe that these findings will aid in sample selection according to the type of analyte and in the further development of standard operating procedures for biobanking.
