Reproducibility and Correlations of Multiplex Cytokine Levels in Asymptomatic Persons

Abstract

Rationale: Cytokines are humoral regulatory molecules that act together in immunologic pathways underlying pathogenesis. Grossly elevated blood levels characterize certain diseases; variations within physiologic ranges could also have significance. We therefore evaluated the performance characteristics of a multiplex cytokine immunoassay. Methods: We used a fluorescent bead-based (Luminex) immunoassay kit to simultaneously measure interleukin (IL) 1B, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12p70, IL13, IFN;, granulocyte colony-stimulating factor, and tumor necrosis factor-A. We tested identical aliquots of serum from 38 asymptomatic individuals on three different days and matched sets of serum, heparinized plasma, and acid citrate dextrose plasma from an additional 38 healthy donors expected to have low cytokine concentrations. We applied multiple imputation to calculate unbiased reproducibility estimates for measurements below the limits of detection. Correlations among the cytokines were assessed by Spearman rank order coefficients and principal components analyses. Results: Of the 13 cytokines, 3 were undetectable (IL1B, IL2, IL5) in more than half of the serum samples. Coefficients of variation for replicate serum measurements ranged from 18% to 44%, with intraclass correlation coefficients ranging from 55% to 98%. Only IL4, IL6, and IL8 had statistically significant correlations (Spearman r, 0.42-0.94) between serum and acid citrate dextrose or heparin plasma levels. Conclusions: Interindividual differences outweigh substantial laboratory variation for these assays, yielding high intraclass correlation coefficients despite unimpressive coefficients of variation. Plasma measurements generally are not reflective of serum levels and hence are not interchangeable. With their small volume, low cost per test, and multiplex capacity, Luminex-based cytokine assays have potential utility for epidemiologic studies. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3450 -6)

Pre‐diagnostic blood immune markers, incidence and progression of B‐cell lymphoma and multiple myeloma: Univariate and functionally informed multivariate analyses

“…Conversely, different media for blood samples (citrate in EPIC‐Italy and EDTA in NSHDS) might have introduced differences in cytokine levels between the two cohorts which may cause bias in unconditional analyses by incomplete correction for cohort status in the model. Although the use of different anticoagulants results in absolute differences in levels of immune markers, correlations between measurements in split samples simultaneously treated with heparin, citrate and EDTA have shown to be highly correlated 15, 30. Similarly, bias may arise from cytokine measurements of study subjects in two phases despite adjustment in multivariate analyses.…”

“…However, several studies have provided evidence of a reasonable between‐to‐within person variability ratio (ICC) suggesting temporal stability for panels of cytokines 30, 31, 32, 33, 34, 35. Finally, blood cytokines are produced not only by those cell types considered to play pivotal roles in the immune system and in inflammatory responses, including lymphocytes, monocytes and mast cells but also by macrophages and, for some cytokines, also fibroblasts, neutrophils and endothelial cells.…”

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

“…The measurements of identical cytokines performed on both platforms were compared using Spearman's coefficient of correlation. The intra-individual serum cytokine variability in the healthy subjects was evaluated using the intra-class correlation coefficient (ICC), which reflects the relative balance of between-and within-person variance components (Agalliu et al, 2013;Clendenen et al, 2010;Dossus et al, 2009;Epstein et al, 2013;Gu et al, 2009;Hardikar et al, 2014;Hofmann et al, 2011;Navarro et al, 2012;Wong et al, 2008). As suggested by Rosner (Rosner, 2000), an ICC ≥ 0.75 indicates excellent reproducibility, 0.4 ≤ ρ <0.75 indicates fair to good reproducibility, and <0.4 indicates poor reproducibility.…”

“…There is no absolute biological reason that would mandate similar and/or correlated concentration of all analytes in sera and plasma samples from the same individual (Altara et al, 2015;Chaturvedi et al, 2011;Clendenen et al, 2010;Dossus et al, 2009;RosenbergHasson et al, 2014;Wong et al, 2008). We therefore determined on each platform the degree of correlation between serum and plasma measurements.…”

Simple Plex™: A Novel Multi‐Analyte, Automated Microfluidic Immunoassay Platform for the Detection of Human and Mouse Cytokines and Chemokines

“…Consequently, multiplexing capability is becoming a critical parameter of biomarker validation and testing as biomarkers are increasingly utilized in all facets of life science research and clinical diagnostics. The adoption of multiplexed testing formats in either clinical research or diagnostics has been severely limited for many reasons, including technical concerns regarding assay reproducibility, cross‐reactivity between the disparate antibody assay components, decreased sensitivity, increased variability, and reported non‐correlation of results with existing methods, including single‐parametric ELISAs 6, 7. As such, many potential multiplex tests continue to be performed as single‐test ELISAs in a parallel processing format.…”