Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays

Wilco de Jager, Katarzyna Bourcier, Ger T Rijkers, Berent J Prakken, Vicki Seyfert-Margolis, Wilco de Jager, Katarzyna Bourcier, Ger T Rijkers, Berent J Prakken, Vicki Seyfert-Margolis

Abstract

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.

Figures

Figure 1
Figure 1
Cytokine profiles in different blood collection tubes. Blood samples were obtained from 4 healthy volunteers using various blood collection tubes after centrifugation cell free plasma (sodium heparin plasma, EDTA plasma and sodium citrate plasma) and serum cytokine levels were measured using a multiplex assay. Color profiles were generated using geometric mean values and plotted using a semi log scale as previously described [14].
Figure 2
Figure 2
Recovery of spiked cytokines in various types of plasma and serum. Cytokines (1000 pg/ml) were spiked in various types of plasma and serum. Recoveries were measured using a multiplex immunoassay. Se serum, NH sodium heparin plasma, E EDTA plasma, CP sodium citrate plasma. Shown are means ± SD.
Figure 3
Figure 3
Long time stability of induced cytokine profiles. Sodium heparin whole blood from 3 individuals was stimulated with a combination of LPS and PHA to induce cytokine profiles. Samples were measured at baseline (BL) and at various time points. Baseline value was set at 100%. Shown are means ± SD.
Figure 4
Figure 4
Recovery of cytokines after multiple freeze - thawing cycle. Aliquots of the same LPS/PHA stimulated samples were freshly thawed or underwent multiple freeze - thawing cycles and cytokines were measured simultaneously. Shown are means ± SD.
Figure 5
Figure 5
Longitudinal assay performance using an internal control sample. Left panel shows variability in time of observed cytokine values of the internal control sample for IL-1β (A), IL-4 (B), I-L6 (C) and TNFα (D) in 50 consecutive runs covering a period of 12 months. Right-hand panel shows overlays of 4 separate standard curves of the corresponding cytokines.

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