title: Reproducibility and correlations of multiplex cytokine levels in asymptomatic persons.
abstract: BACKGROUND
  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) 1beta, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12p70, IL13, IFNgamma, granulocyte colony-stimulating factor, and tumor necrosis factor-alpha. 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 (IL1beta, 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 rho, 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.
authors.affiliation: Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, 6120 Executive Boulevard, Room 7073, Rockville, MD 20852, USA. wongh@mail.nih.gov
authors.forename: Hui-Lee
authors.initials: HL
authors.lastname: Wong
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Pfeiffer
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Fears
authors.forename: Roel
authors.initials: R
authors.lastname: Vermeulen
authors.forename: Shaoquan
authors.initials: S
authors.lastname: Ji
authors.forename: Charles S
authors.initials: CS
authors.lastname: Rabkin
mesh_terms: D016207:Cytokines / Q000097:blood*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D008297:Male; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results
chemical_list: D016207:Cytokines; D011933:Reagent Kits, Diagnostic