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Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays

Wilco de Jager123*, Katarzyna Bourcier4, Ger T Rijkers35, Berent J Prakken123 and Vicki Seyfert-Margolis4

Author Affiliations

1 Department of Pediatric Immunology, Centre for Molecular and Cellular Intervention (CMCI), University Medical Centre Utrecht, Utrecht, The Netherlands

2 EUREKA Institute of Translational Medicine, Siracusa, Italy

3 Immune Tolerance Network, Luminex Core Facility, Utrecht, the Netherlands

4 Immune Tolerance Network, University of California, San Francisco, CA, Bethesda, MD, USA

5 Laboratory of Medical Microbiology and Immunology, Sint Antonius Hospital, Nieuwegein, the Netherlands

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BMC Immunology 2009, 10:52  doi:10.1186/1471-2172-10-52

Published: 28 September 2009



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.


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°C. After 4 years several cytokines (IL-1α, IL-1β, 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.


All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.