Biomarker Assay Procedures for TNFα System

Fasting blood samples were centrifuged for 10 min at 4°C, aliquoted in 0.5–1 mL volumes of EDTA plasma (serum for CRP), and stored at −80°C within 2 h of venipuncture. Frozen samples from the Jackson field center were shipped to the Mayo Clinic Immunochemical Core Laboratory (Rochester, MN) overnight on dry ice. Samples were thawed on ice, repackaged into Eppendorf tubes with bar codes and refrozen for shipping to SearchLight^™ (ThermoScientific). CRP assays were performed using immunoturbidometric assays (Diasorin, Inc, Stillwater, MN; inter-assay imprecision 1.8–2.6%; intra-assay imprecision 1.0–9.2%) and multiplex assays (SearchLight™, Pierce, Boston, MA) were used for IL-6 and sTNFRs. sTNFR fractions show stability over time with longer half-lives than TNFα levels and have been validated as sensitive indicators of TNFα system activation^{34 (link)}. Precision of the assays performed by SearchLight^TM was retrospectively determined based on data derived from a blinded, internal plasma control sample. Algorithms were developed to reduce plate-to-plate variations in protein levels and all analyses used these normalized data.^{35 (link)}