Validation of two multiplex platforms to quantify circulating markers of inflammation and endothelial injury in severe infection

Abstract

Biomarkers can prognosticate outcome and enable risk-stratification. In severe infection, focusing on multiple markers reflecting pathophysiological mechanisms of organ injury could enhance management and pathway-directed therapeutics. Limited data exist on the performance of multiplex biomarker platforms. Our goal was to compare endothelial and immune activation biomarkers in severe pediatric infections using two multiplex platforms. Frozen plasma from 410 children presenting to the Jinja Regional Hospital in Uganda with suspected infection was used to measure biomarkers of endothelial (Angiopoietin-2, sFlt-1, sVCAM-1, sICAM-1) and immune (IL-6, IP-10, sTNFR-1, CHI3L1) activation. Two multiplex platforms (Luminex®, EllaTM) based on monoclonal antibody sandwich immunoassays using biotin-streptavidin conjugate chemistry were selected with reagents from R&D Systems. The two platforms differed in ease and time of completion, number of samples per assay, and dynamic concentration range. Intra-assay variability assessed using a coefficient of variation (CV%) was 2.2–3.4 for Luminex® and 1.2–2.9 for EllaTM. Correlations for biomarker concentrations within dynamic range of both platforms were best for IL-6 (ρ = 0.96, p<0.0001), IP-10 (ρ = 0.94, p<0.0001) and sFlt-1 (ρ = 0.94, p<0.0001). Agreement between concentrations obtained by both methods assessed by the Bland-Altman test varied, with best agreement for CHI3L1. Our data suggest that biomarkers of endothelial and immune activation can be readily measured with multiplex platforms. Luminex® and EllaTM produced reliable results with excellent CV% values. The EllaTM platform was more automated and completed in 75 minutes, potentially compatible with near-patient use. Trends in concentrations obtained by these methods were highly correlated, although absolute values varied, suggesting caution is required when comparing data from different multiplex platforms.

Biomarkers of endothelial activation/dysfunction distinguish subgroups of Ugandan patients with sepsis and differing mortality risks

“…The clinical samples were centrifuged at the hospital, and serum was stored at -20°C. Commercial ELISAs were used to measure biomarker levels (ANGPT1, ANGPT2, sICAM1, sTREM1, CHI3L1, PF4, CXCL10, sFLT1, sTEK, VEGF: R&D Systems; vWF: antibody from Dako, standard from American Diagnostica) (38). All assays were conducted in duplicate.…”

Plasma APRIL as a Potential Biomarker in the Diagnosis of Obstructive Sleep Apnea

“…Magnetic Luminex® Assays was a magnetic bead-based antibody microarray founded upon the sandwich immunoassay principle, which can be used to facilitate the simultaneous quantitation of up to 100 soluble analytes in a single sample [29]. APRIL, MCP-1 and L-selectin concentrations were determined by the Luminex magnetic bead cytokines panel based on the competition principle (R&D Systems, Inc. Minneapolis, MN, USA).…”

Risk-stratification of febrile African children at risk of sepsis using sTREM-1 as basis for a rapid triage test

“…Eleven of 13 biomarkers of endothelial or immune activation 6− 13 used in our previously reported study in febrile adults 14 were quanti ed using the multiplex Luminex® platform (Luminex, Austin TX) with custom-developed reagents (R&D Systems, Minneapolis, MN) 39 in the 2084 children with available plasma who had been regularly discharged from hospital or had died (see Supplementary Information). sTREM-1 and sFlt-1, which ranked rst and second in the analysis of the derivation cohort, sTNFR1, which ranked second after sTREM-1 in the previously reported study in febrile adults in Tanzania, 14 and Ang-2, which was the biomarker used for sample size considerations of the current study, were subsequently quanti ed using identical methods in the remaining 376 children with available plasma who were transferred or absconded.…”