Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays.
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.


CONCLUSIONS
All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.
Department of Pediatric Immunology, Centre for Molecular and Cellular Intervention (CMCI), University Medical Centre Utrecht, Utrecht, The Netherlands. wjager@umcutrecht.nl
Wilco
W
de Jager
Katarzyna
K
Bourcier
Ger T
GT
Rijkers
Berent J
BJ
Prakken
Vicki
V
Seyfert-Margolis
D001800:Blood Specimen Collection / Q000009:adverse effects; D002478:Cells, Cultured; D002986:Clinical Trials as Topic; D016207:Cytokines / Q000378:metabolism*; D004492:Edetic Acid / Q000378:metabolism; D005615:Freezing / Q000009:adverse effects; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods / Q000592:standards; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008070:Lipopolysaccharides / Q000378:metabolism; D010835:Phytohemagglutinins / Q000378:metabolism; D055550:Protein Stability; D012015:Reference Standards; D015203:Reproducibility of Results
D016207:Cytokines; D008070:Lipopolysaccharides; D010835:Phytohemagglutinins; D006493:Heparin; D004492:Edetic Acid