Two Methods for Evaluating the Effect of pre-Analytical Handling Procedures on Interleukin 6, Interleukin 8, and Tumor Necrosis Factor α.

Abstract

Background: Except host and environmental factors influencing individual human cytokine responses, pre-analytical handling procedures and detection methods also affect cytokine levels.

Methods: In this study, we used cytometric bead array (CBA) and chemiluminescence (ECL). These two methods were used to test serum and plasma samples from 50 healthy adult volunteers and 50 rheumatoid arthritis (RA) patients' cytokine levels. We evaluated the impact of storage temperature, collection times, and additives in collection tubes on the measurement of IL-6, IL-8, and TNF-α.

Results: The finding of this study first indicated that the CBA assay for IL-6 and IL-8 showed excellent agreement with ECL for the same analysis, but the CBA assay and ECL for TNF-α measurement showed less agreement. Furthermore, we used two detection methods to find plasma cytokines showing more stability than serum cytokines, when the samples were stored at 4°C. Additionally, IL-8 concentration was affected by the storage conditions of whole blood from the time of collection until further processing at room temperature. We also found heparin tubes showed higher levels of IL-6 and TNF-α than other tubes at room temperature.

Conclusions: In general, the best values for IL-6, IL-8, and TNF-α were found in EDTA samples, stored at 4°C, and centrifuged quickly within 2 hours. The effect of pre-analytical handling procedures and detection methods on cytokine levels we identified would provide a basis for clinical cytokine detection.