Rationale Cytokines are mutually interdependent regulatory molecules that act together in humoral networks characterized by redundancy and antagonism. However, the performance of available multiplex assays on physiologic cytokine levels has not been characterized. We therefore evaluated reproducibility and effect of blood processing for a fluorescent bead-based multiplex (Luminex) cytokine immunoassay. Methods We measured levels of interleukin-(IL)1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL13, interferon-(IFN)γ, granulocyte colony-stimulating factor (GMCS-F) and tumor necrosis factor-(TNF)α in 50 uL serum or plasma samples with the LINCOplex® 13-plex Cytokine Kit (Millipore, Billerica, MA). Reproducibility was assessed by testing serum samples from 38 asymptomatic individuals on three different days. To assess the effects of blood processing, we compared cytokine concentrations in matched sets of serum, acid citrate dextrose (ACD) anticoagulated plasma, and heparinized plasma from an additional 38 blood donors. We used multiple imputation for measurements below the limits of detection to calculate unbiased estimates of reproducibility. Results Of the 13 cytokines, three (IL1β, IL2, IL5) were undetectable in more than half of the serum samples. Replicate serum measurements of the various cytokines had coefficients of variation (CV) ranging from 18-44% and intraclass correlation (ICC) ranging from 0.81-0.98. Only IL4, IL6 and IL8 had significant correlation between serum and plasma levels (Spearman ρ range: 0.42-0.94), with both ACD and heparin providing similar results. Conclusions The wide variability in cytokine levels between individuals outweighs the substantial laboratory variation in these assays. Thus, despite unimpressive CVs, we found high ICCs, implying potential utility for case-control and prospective epidemiologic studies. Plasma measurements do not generally correspond to serum levels and hence, are not interchangeable. Our data also do not address the validity of cytokine measurements that may be affected by cross-reactivity, temporal fluctuations and/or other artifacts. However, with the low sample volume requirement, low assay cost per analyte and potential for many analytes per sample, Luminex-based cytokine assays may help elucidate immunologic pathways in pathogenesis.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA