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Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays
Wilco de Jager1, Katarzyna Bourcier, Ger T Rijkers
1Department of Pediatric Immunology, Centre for Molecular and Cellular Intervention (CMCI), University Medical Centre Utrecht, Utrecht, The Netherlands. wjager@umcutrecht.nl
Insights
Proper sample handling and storage are crucial for reliable cytokine measurements in clinical trials. Cytokines are stable for up to 2 years at -80°C, but degrade after 4 years and are sensitive to freeze-thaw cycles.
Area of Science:
- Immunology
- Biochemistry
Background:
- Advances in understanding immune cellular interactions and cytokine networks have spurred the development of monoclonal antibody therapies.
- Systemic cytokine levels serve as vital surrogate markers for evaluating inflammatory pathways in vivo during therapeutic interventions.
- Multiplex immunoassay (MIA) technologies enable the simultaneous detection of multiple cytokines, but sample handling and storage can impact assay reliability.
Purpose of the Study:
- To investigate the impact of pre-analytical variables on cytokine detection using multiplex immunoassays.
- To assess the influence of blood collection tubes, storage duration, and freeze-thaw cycles on cytokine profiles.
Main Methods:
- Cytokine recovery was assessed by spiking known amounts into plasma and serum samples.
- Stability of cytokines was evaluated during long-term storage at -80°C and after multiple freeze-thaw cycles.
- An internal control was developed and implemented for multiplex cytokine immunoassays.
Main Results:
- Cytokine recoveries generally ranged from 80-120% in spiked samples.
- Cytokines remained stable for up to 2 years of storage at -80°C.
- Significant degradation (up to 75%) was observed for several cytokines (IL-1α, IL-1β, IL-10, IL-15, CXCL8) after 4 years of storage. Only 2 out of 15 cytokines were stable after multiple freeze-thaw cycles.
Conclusions:
- Key parameters influencing cytokine measurement reliability in clinical trials were identified.
- Establishing standardized protocols for sample handling, storage, and processing is essential for accurate cytokine analysis.
Background:
Growing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles.
Results:
Since under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80 degrees C. After 4 years several cytokines (IL-1alpha, IL-1beta, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays.
Conclusion:
All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.
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