Quickplex sq 120

To avoid a sample selection bias, the parents or legal guardians of eligible infants were approached as early as possible after birth (usually <72 h). To limit invasiveness, the research was analyzed only from the residual (leftover) serum of weekly routine testing from the hospital laboratory. Serum cytokines were detected on Days 1–3, Days 7–14, and Days 21–28 after birth. Our study detected ten cytokines, including IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNF-α. To minimize protein degradation, strict protocols were employed in our hospital laboratory. Blood collected in BD Microtainers (Becton Dickinson, Mississauga, Ontario, Canada) was spun down within 1 h to remove the cell fraction, and the leftover serum was immediately stored at −80°C. The serum was collected to detect the cytokine protein level via the MSD technology using the U-PLEX Biomarker Group 1 (Human) Multiplex Assay (K15049D). A MESO QuickPlex SQ 120 machine and MSD Discovery Workbench version 4.0.12 were used for detection and data analysis, respectively.

At baseline and week 4, we analysed serum high-sensitivity (hs) CRP using a Roche Cobas 8000 (Mannheim, Germany) (von Eckardstein et al., 2013 (link)). Serum pro-inflammatory and anti-inflammatory cytokines, including IFN-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and tumour necrosis factor alpha (TNF)-α were measured using Meso Scale Discovery (MSD) V-PLEX sandwich immunoassays, MSD Pro-inflammatory Panel 1 (human) kit (Dabitao et al., 2011 (link); King et al., 2019 (link)) and plates read on an MSD QuickPlex SQ 120 (Rockville, Maryland, USA), as previously published (Hepgul et al., 2012 (link); Russell et al., 2019 (link)). The inter-assay coefficient of variations was <10%. The results were analysed using MSD DISCOVERY WORKBENCH analysis software. Of note, levels of IL-1β, IL-4 and IL-12p70 were below the minimum detectable value for most of the subjects, so these cytokines were not included in the statistical analyses.

The concentration of the selected eight cytokines (IFN‐γ, IL‐10, IL‐17A, IL‐1β, IL‐4, IL‐5, IL‐6, and TNF‐α) was measured in undiluted plasma samples of the three subjects using a multiplex MSD (Mesoscale Discoveries) U‐plex Biomarker Group 1 (human) assay (MSD, K15067L) in the MSD MESO QuickPlex SQ 120 platform following the manufacturer’s instructions.

Venous blood was collected and spun down to obtain serum using standard clinical practices. Serum IL6 and TNFα were determined using a Meso Scale Discovery (MSD; Rockville, MD) Quick Plex SQ 120 imager using electrochemiluminescence technology. Minimum sensitivity for the IL6 assay was 0.07 pg/mL, while sensitivity was 0.09 pg/mL for TNFα. Intra-assay coefficients of variation (CV) were 7.84 and 7.67%, and inter-assay coefficients were 5.78 and 2.5% for IL6 and TNFα, respectively. IGF1 was assessed via immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX). The inter-assay CV, intra-assay CV, and assay sensitivity for IGF1 were 9.43, 3.48, and 4.89 ng/mL, respectively.