Clinical evaluation of the levels of 12 cytokines in serum/plasma under various storage conditions using evidence biochip arrays.

Cytokines are a group of peptides which form a sophisticated network to modulate multiple cellular events. Within such a network, and through complex feedback mechanisms, cytokine functions are largely interdependent and closely associated with a number of pathological processes. In the present study, the EVIDENCE 180 system was used to study the effects of storage temperature and repeated freeze/thaw cycles on the concentration of 12 cytokines in various sample types. Samples were collected from 9 healthy volunteers and stored by 3 methods: gel, glass and lithium heparin (LH) tubes. Immediately following collection, the concentration of each cytokine in the samples was measured. Cytokine concentrations of the 3 sample types that did not undergo repeated freeze/thaw cycles were compared with those subjected to 1‑10 freeze/thaw cycles. In addition, the dynamic changes of 6 sample types which were stored at 4˚C for 6 h to 6 days was analyzed. In addition, the within‑ and between‑run precision of 12 cytokines on the biochip array was evaluated. Interleukin (IL)‑8, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) concentrations were lower in plasma compared with serum. Cytokine levels in serum and plasma were affected by several freeze/thaw cycles with IL‑1β, ‑4 and ‑10 increasing significantly following 1 freeze/thaw cycle and remaining at stable increased levels for the duration of the additional 9 cycles. In separated serum samples in gel and glass tubes stored at 4˚C for 6 days, no difference in concentration of the 12 cytokines was identified. In the other 4 sample types, IL‑8, VEGF, tumor necrosis factor α and EGF levels were altered when stored at 4˚C. Results indicate that the EVIDENCE 180 system is stable and plasma was observed as the best sample type to determine concentration of the 12 cytokines using this biochip array. Repeated freeze/thaw cycles and storage at 4˚C was identified to affect the concentration of the 12 cytokines. The current study demonstrates that repeated freeze/thaw cycles of samples must be avoid. In addition, results indicate that plasma or serum must be separated immediately following centrifugation and sample concentration should be measured as soon as possible.