Fluorokine map

WBC count and differential was determined by the clinical laboratory. Classification of abnormal cell counts were based on the laboratory’s normative data as follows: WBCs>10,000/μL, PMNs >7,000/μL, lymphocytes <1,000/μL, monocytes >800/μL and eosinophils <50/μL (there is no universally accepted lower limit for eosinophils, this number is thus somewhat arbitrary). The concentrations of ACTH and cortisol were also determined by the clinical laboratory using standard methodologies. Interleukin (IL)-6, was measured with a cytometric bead-based system (Fluorokine MAP; R&D Systems); the lower limit of detection was 1.1 pg/mL. Standard enzyme linked immunoassay (ELISA) was used to determine plasma concentrations of plasma metanephrines (IBL-America; sensitivity = 14.9 pg/mL).

Two electrochemiluminesence-based multiplex assay panels (Proinflammatory 9-plex and Chemokine 7-plex; Meso-Scale Diagnostics, LLC, Rockville, MD) were used to determine concentrations of IL-1β, IL-2, IL-6, IL-10, IL-12p70, GM-CSF, IFN-γ, TNF-α, CXCL8 (IL-8), CXCL10 (IP-10), CCL11 (eotaxin), CCL2 (MCP-1), CCL13 (MCP-4), CCL4 (MIP-1β), and CCL17 (TARC). All testing was done at a centralized laboratory. Analyte- and plate-specific lower limits of detection (LLOD) were calculated as concentrations 2.5 standard deviations above the background for each analyte on each plate. Concentrations of five soluble receptors (sCD14, sgp130, sIL-2Rα, sTNF-R2), a cytokine (BAFF), and a chemokine CXCL13 (BLC-BCA1), were measured in a single panel (Human Biomarker Custom Premix Kit A) using the fluorescent bead-based multiplexed Luminex xMAP system at a centralized laboratory (Fluorokine^® MAP, R&D Systems, Minneapolis, MN), and a Bio-Plex 200 Luminex instrument and Bio-Plex software (Bio-Rad, Hercules, CA). A single assay lot was used. Finally, CRP was measured at Quest Diagnostics using a high-sensitivity nephelometric assay (Dade Behring, Inc., Newark, DE). All specimens for any given individual were run on the same plate.

The methods utilized to assay soluble CD14 in plasma have been reported in detail elsewhere [31 (link)]. Briefly, plasma soluble CD14 concentration was measured at the University of California, Los Angeles, as one of several analytes tested with multiplexed (Luminex platform) assay kits (Fluorokine MAP, R&D Systems, Minneapolis, MN, used according to manufacturer directions), a Bio-Plex 200 Luminex instrument and Bio-plex analysis software (Bio-Rad, Hercules, CA). Samples from matched case-control pairs were tested in the same batch, with the order within pairs determined at random; pairs of quality control (QC) samples resembling pairs of study samples (amounting to approximately 10% of samples) were randomly inserted into each batch to monitor assay performance. Laboratory personnel were blinded to the samples’ case/control status and the identity of QC specimens. Additionally, batch correction using Rosner et al.‘s method was implemented to minimize the potential influence of laboratory batch-related variability [35 (link)].

Serum concentrations of the soluble receptors were determined using the multiplexed Luminex xMAP system (Fluorokine® MAP) using assays produced by R & D systems (Minneapolis, MN) following the manufacturer’s instructions, and a Bio-Plex 200 Luminex instrument and Bio-Plex software (Bio-Rad, Hercules, CA). Concentrations of four soluble receptors (sCD14, sgp130, sIL-2Rα, sTNF-R2), plus a cytokine (BAFF) and the chemokine CXCL13, were measured in a single panel (Human Biomarker Custom Premix Kit A). All testing for these markers was conducted in a single laboratory at the University of California, Los Angeles. One external serum control from a normal donor (no spiked values) was run in duplicate on each assay. This control sample was from a single blood draw that was aliquoted multiply and stored at − 80°C. For each plate tested, a biomarker- and plate-specific LLOD was defined. All the samples tested in the multiplex Kit A had concentrations above the lowest standard of the standard curve; therefore, the LLOD was defined as the observed concentration of the lowest standard.