Stability of interleukin-1β, -4, -6, -8, -10, -13, interferon-γ and tumor necrosis factor-α in human sera after repetitive freeze-thaw cycles and long storage

Cytokines (CKs) are one of the key components of the molecular network modulating multiple immunological interactions. Within such biological systems, CKs functions are associated with several processes, thus quantification of these analytes in serum samples, as well as a faithful determination of its concentration, are crucial for the translational aspect of many studies. This study is focused on the evaluation of the effects of storage duration and multiple freeze-thaw cycles on CKs stability. Serum samples were obtained from 24 healthy participants. Samples were prospectively stored at 4 °C for 1–7 and 30 days, and also underwent multiple freeze-thaw cycles. Afterwards, CK levels were determined by enzyme-linked immunosorbent assay. Among the 8 examined CKs all of them showed significant degradation (determined with the two-way ANOVA and post-hoc test) after 4 days of sample storage at 4 °C. Serum were affected by freezing at −20 °C and thawing, and 2 of CKs (IL-1β and IL-8) showed significant concentration decrease after following 2 freeze-thaw cycles. It has been also determineded that CKs in serum samples after multiple freeze-thaw cycles had better stability, when samples were stored at −80 °C (compared to storage at −20 °C). This study demonstrates that long storage at 4 °C, as well as multiple freeze-thaw cycles of serum samples, must be avoided and CK concentrations should be measured immediately after sample collection.