title: Multiplex serum biomarker assessments: technical and biostatistical issues.
abstract: BACKGROUND
  Identification of predictive and prognostic biomarkers for patients with disease and undergoing different therapeutic options is a very active area of investigation. Many of these studies seek biomarkers among circulating proteins accessed in blood. Many levels of standardization in materials and procedures have been identified which can impact the resulting data.
  
  
  METHODS
  Here, we have observed unexpected variability in levels of commonly tested analytes in serum which were processed and stored under standardized conditions. We have identified apparent changes in cytokine, chemokine and growth factor levels detected by multiplex Luminex assay in melanoma patient and healthy donor serum samples, over storage time at -80°C. Controls included Luminex kit standards, multiplexed cytokine standards and WHO cytokine controls. Data were analyzed by Wilcoxon rank-sum testing and Spearman's test for correlations.
  
  
  RESULTS
  The interpretation of these changes is confounded by lot-to-lot kit standard curve reagent changes made by a single manufacturer of Luminex kits.
  
  
  CONCLUSIONS
  This study identifies previously unknown sources of variation in a commonly used biomarker assay, and suggests additional levels of controls needed for identification of true changes in circulating protein levels.
authors: University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA. butterfieldl@upmc.edu
authors: Lisa H
authors: LH
authors: Butterfield
authors: Douglas M
authors: DM
authors: Potter
authors: John M
authors: JM
authors: Kirkwood
mesh_terms: D015415:Biomarkers / Q000097:blood*; D056808:Biostatistics / Q000379:methods*; D001782:Blood Donors; D002624:Chemistry, Clinical / Q000379:methods*; D018925:Chemokines / Q000097:blood; D006262:Health; D006801:Humans; D057230:Limit of Detection; D008545:Melanoma / Q000097:blood; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D015203:Reproducibility of Results; D013997:Time Factors; D014944:World Health Organization
chemical_list: D015415:Biomarkers; D018925:Chemokines; D011933:Reagent Kits, Diagnostic