Stability of 12 T-helper cell-associated cytokines in human serum under different pre-analytical conditions

Cytokines are soluble and readily analyzed signaling molecules which reveal vital cues about the state of the immune system. As such, they serve in diagnosis and monitoring of immune-related disorders, where strictly controlled handling of the samples including storage and freeze/thawing procedures are required. In basic research and clinical trials, human serum samples can be left for long-term storage before processing. Storage space is commonly limited in scientific laboratories, which require storage of fewer but larger aliquots of patient serum samples. There are also practical limitations to the number of analytes to be processed at the same time. Further, new findings and technological progress might prompt analysis of hitherto unconsidered or undetectable molecules. Repeated freeze/thawing of serum samples is therefore a likely scenario, raising the question of the stability of the measured analytes under such conditions. To address this question, we subjected serum samples with spiked-in T-helper cell associated cytokines to several cycles of freeze/thawing under different conditions, including storage at -20 degrees C or -80 degrees C and thawing at 4 degrees C, 22 degrees C, and 37 degrees C, respectively. The concentration of TNF-alpha, IL-4, IL-17F, and IL-22 decreased after storage at room temperature for 4 h before freezing. Generally, storage at -20 degrees C resulted in reduced cytokine concentrations. This contrasts storage at -80 degrees C, which gave stable analyte concentrations; unaffected by repeated freeze/thaw cycles. The study presented here highlights the need for sentinel samples with known cytokine concentrations as internal control for the freeze/thaw process.