V plex human biomarker 54 plex kit

Levels of 54 serum proteins were quantified using a V-plex Human Biomarker 54-plex kit (Meso Scale Diagnostics), as per manufacturer's recommendations. Proteins analyzed included; CRP, Eotaxin, Eotaxin-3, FGF, Flt-1/VEGFR-1, GM-CSF, ICAM-1, IFN-γ, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-1RA, IL-1α, IL-1β, IL-2, IL-21, IL-22, IL-23, IL-27, IL-3, IL-31, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IL-9, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α, PlGF, SAA, TARC, Tie-2, TNF-α, TNF-β, TSLP, VCAM-1, VEGF-A, VEGF-C, VEGF-D. Levels of circulating proteins were compared to percentage MAIT cell data available for n = 26 OAC patient blood samples, n = 16 OAC tumor samples, and a subset of n = 12 OAC patients for whom both matched blood and tumor MAIT cell percentages were available, and Spearman (r) correlation values were calculated using GraphPad Prism (Version 5). Correlation values of >0.6/<-0.6 were considered strongly correlated. Analytes below the kit detection range were not included.

Approximately 2 mL of whole blood was collected from each study participant and partitioned via centrifugation (for 10 min at 1960 g) for the isolation of plasma, peripheral blood mononuclear cells, and serum. Plasma was then carefully separated, avoiding the buffy coat, and transferred into separate tubes to create 1 mL aliquots. Next, the aliquots were frozen slowly at a temperature of − 80 °C for 24 to 72 h and transferred into the liquid nitrogen vapor phase for storage.
Plasma samples were analyzed for the presence of 54 proinflammatory markers including cytokines, chemokines, Th17 mediators, angiogenetic and vascular injury factors using the V-PLEX Human Biomarker 54-Plex Kit (MesoScale Diagnostics, Rockville, MD) Human Biomarker 54-Plex Kit consisting of Proinflammatory Panel 1, Cytokine Panel 1, Cytokine Panel 2, Chemokine Panel 1, Th17 Panel 1, Angiogenesis Panel 1 and the Vascular Injury Panel 2 according to the manufacturer’s instructions. All samples were run in two batches of 54-plex kits. 8 random technical duplicates were used to extrapolate the average CV. The presence of the analytes was measured in 96 well plates using the Quickplex SQ 120 instrument (Mesoscale) via electrochemiluminescence. Sample concentrations were calculated following extrapolation of the analyte standard curves with a four-parameter logistic fit using MSD Workbench 3.0 software.

Peripheral blood was collected into BD Vacutainer K2 EDTA tubes (Beckton Dickinson) and separated into plasma, buffy coat and red blood cells (RBCs). CRP and IL6 were measured in two technical replicates for each plasma sample using V-PLEX Human Biomarker 54-Plex Kit ((Meso Scale Discovery (MSD)) on a MESO QuickPlex SQ 120 instrument. Concentration values (pg ml⁻¹) were calculated against a standard curve using provided calibrators and imported to R v4.0.1. For each analyte, missing values were replaced with either the minimum (if below-fit curve range) or maximum (if above-fit curve range) calculated concentration and means of replicates used in downstream analysis. Extreme outliers were classified as measurements more than three times the interquartile range below or above the first and third quartiles, respectively, and excluded from the analysis. Differential abundance was defined using mixed effects linear regression as implemented in the lmer() function from the lmerTest R package v3.1-2 with log₂-transformed concentration as the outcome/dependent variable, trisomy 21 status as the predictor/independent variable, age and sex as fixed covariates and sample source as a random effect. Multiple hypothesis correction was performed with the BH method using FDR10% (q < 0.1).