Biomarker Determinations
inflammation, tumor necrosis
INFLAMMATION, TUMOR NECROSIS, SENSITIVITY, NEUROLOGICAL DISORDERS
A sample size of 86 neonates per group is required to detect a 0.5-SD difference on the log scale with a single observation per neonate, with 80% power while controlling for a type I error of .0167 (3 markers, Bonferroni). Whole blood specimens (1.5 mL) were collected in heparinized tubes at 3 timed intervals: (1) baseline (median [IQR] 17.0 [12.9-21.8] hours), (2) day 2, drawn 24 ± 2 hours after the first study dose was given, and (3) day 4, 72 ± 2 hours after the first study drug dose. Specimens were centrifuged at 6000 rpm for 10 minutes and plasma collected and stored at −80 °C within 4 hours of collection.Biomarkers of inflammation included both pro-inflammatory and anti-inflammatory mediators. The MILLIPLEX MAP Human High Sensitivity T Cell Panel from Millipore-Sigma was used to measure 12 analytes: Interleukin (IL) 1b, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, macrophage inflammatory protein (MIP)–1a/chemokine ligand (CCL) 3, MIP-1b/CCL4, interferon (IFN)-γ, fractalkine/CX3CL1, and tumor necrosis factor (TNF)–α. This high sensitivity panel uses Luminex xMAP technology that allows for multiplex analyte detection with adequate recovery rates in children with neurological disorders.All assays were performed in duplicate, and results were averaged for analyses. All assays were performed according to the manufacturers’ recommended protocols.