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Updated: Dec 27, 2025
Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety
Published on: September 15, 2023
Stability of 12 T-helper cell-associated cytokines in human serum under different pre-analytical conditions
Ulrik Stervbo1, Sharon Bajda2, Patrizia Wehler2
1Center for Translational Medicine, Medical Department I, Marien Hospital Herne, University Hospital of the Ruhr-University Bochum, Hölkeskampring 40, 44625 Herne, Germany; Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin-Brandenburg Center for Regenerative Therapies, Augustenburger Platz 1, 13353 Berlin, Germany.
Insights
Repeatedly freezing and thawing human serum samples impacts cytokine stability. Storing samples at -80°C preserves cytokine concentrations, unlike -20°C or room temperature conditions, ensuring reliable immune system analysis.
Area of Science:
- Immunology
- Biochemistry
- Laboratory Medicine
Background:
- Cytokines are crucial immune system signaling molecules used in diagnosing and monitoring immune disorders.
- Proper sample handling, including storage and freeze-thaw cycles, is vital for accurate cytokine analysis.
- Limited laboratory space and processing capabilities necessitate long-term serum sample storage and potential for repeated freeze-thaw cycles.
Purpose of the Study:
- To investigate the stability of T-helper cell associated cytokines in human serum samples subjected to repeated freeze-thaw cycles under various storage and thawing conditions.
- To determine optimal storage and thawing protocols to maintain cytokine integrity for reliable diagnostic and research applications.
Main Methods:
- Human serum samples were spiked with specific cytokines (TNF-α, IL-4, IL-17F, IL-22).
- Samples underwent multiple freeze-thaw cycles after storage at -20°C or -80°C.
- Thawing occurred at different temperatures (4°C, 22°C, 37°C).
- Cytokine concentrations were measured to assess stability.
Main Results:
- Cytokine concentrations (TNF-α, IL-4, IL-17F, IL-22) decreased after room temperature storage for 4 hours before freezing.
- Storage at -20°C generally led to reduced cytokine concentrations.
- -80°C storage maintained stable analyte concentrations, unaffected by repeated freeze-thaw cycles.
Conclusions:
- Serum sample storage at -80°C is recommended to preserve cytokine stability during repeated freeze-thaw cycles.
- Storage at -20°C or exposure to room temperature before freezing can compromise cytokine integrity.
- Utilizing sentinel samples with known cytokine concentrations is advised as an internal control for freeze-thaw processes.
Abstract:
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 °C or -80 °C and thawing at 4 °C, 22 °C, and 37 °C, respectively. The concentration of TNF-α, IL-4, IL-17F, and IL-22 decreased after storage at room temperature for 4 h before freezing. Generally, storage at -20 °C resulted in reduced cytokine concentrations. This contrasts storage at -80 °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.
