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

Date Published: April 18, 2017

Publisher: Public Library of Science

Author(s): Aleksandra Leligdowicz, Andrea L. Conroy, Michael Hawkes, Kathleen Zhong, Gerald Lebovic, Michael A. Matthay, Kevin C. Kain, Viswanathan V. Krishnan.


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.

Partial Text

The search for novel biological markers to predict response to therapies, prognosticate outcome, or assist in patient enrollment in clinical therapeutic trials is quickly evolving [1]. In the context of life-threatening infection, many biomarkers have been proposed to improve the discriminatory ability to achieve these goals [2, 3]. Emphasizing markers of pathophysiological pathways involved in severe infections and focusing on multiplex platforms with near-patient or point-of-care potential, could accelerate the development of precision medicine tools for life-threatening infections [4, 5].

In the past decade, the use of multiplex immunoassays has made the quantification of multiple analytes obtained from clinical samples more feasible. However the variety of potential platforms and reagents has created challenges in selecting optimal assays for large-scale projects that demand consistent results and ultimately has hindered the use of these platforms in clinical research and their translation into clinical practice [25, 27, 40]. The lack of available data comparing multiplex platforms using consistent reagents makes it difficult to compare previous studies examining host biomarkers of severe infections. In this study, we evaluated two different multiplex platforms (Luminex® and EllaTM) using reagents from a single manufacturer (R&D Systems) to test over 400 well-annotated plasma samples from febrile children presenting with suspected severe infections. We found that the correlations between values for biomarkers of endothelial and immune activation tested within assay dynamic range were excellent. Moreover, using two different statistical methods, we identified consistent differences between absolute biomarker concentrations obtained using the two different multiplex platforms.




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