Bio plex pro human panels

Nineteen inflammatory biomarkers were measured in the third trimester (mean [SD] gestational age = 33.1 [1.5] weeks) plasma samples (Table 1 and Table S1; http://links.lww.com/EE/A233). Maternal whole blood was treated with ethylenediaminetetraacetic acid (EDTA) and a serine protease inhibitor, phenylmethylsulfonyl fluoride, then clarified to obtain plasma. MMPs, CRP, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), IL-2, 6, 8, 12, and tumor necrosis factor alpha (TNF-α) were measured using Milliplex Map kits (Millipore, Canada). Vascular cell adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), vascular endothelial growth factor (VEGF), MCP-1, and macrophage inflammatory protein-1 beta (MIP-1β) were measured using Bio-Plex Pro Human panels (Bio-Rad, Canada). Laboratory analyses were conducted by the Environmental Health Science and Research Bureau at Health Canada. Intra and interassay coefficients of variation for all biomarkers were below 11% and 20%, respectively.^{25 (link)}Our primary outcome was a composite index of proinflammatory biomarkers using knowledge of biomarker function.^{25 (link)} This index was derived by summing the standardized z-scores of MIP-1β, TNF-α, IFN-γ, MCP-1, IL-2, IL-6, IL-8, and IL-12 using the within sample means and SDs to calculate each z-score.

Analysis of human plasma samples for acute phase proteins relevant to cardiovascular diseases [C-reactive protein (CRP), haptoglobin, fibrinogen, platelet factor (PF4), adiponectin, vonWillebrand Factor (vWF), α₂-macroglobulin (A2M), α-acid glycoprotein (AGP), serum amyloid protein (SAP), L-selectin)], and cytokines [interleukins (IL-1, − 2,-4,-5,-6,-7, − 8,-10, − 12, − 13), tumour necrosis factor (TNF-α), granulocyte macrophage colony-stimulating factor (GMCSF) and interferon gamma (IFN-γ)] were done by affinity-based multiplex protein array assays using Bio-Plex Pro Human panels (Biorad) and Milliplex Map kits (Millipore) with a Bioplex 100 instrument (Biorad), based on the procedure reported by Kumarathasan et al., 2014 [37 (link)].

CRP was measured in all women who had live, singleton births and provided a blood sample in their third trimester (n = 1,573). The average weeks at gestation for the blood draw was 31.6. CRP was analyzed through an affinity-based multiplex protein array, with Bio-Plex Pro Human panels (Bio-Rad, ON, Canada) and Milliplex Map kits (Millipore, Billerica, MA).^{56 (link)} The interassay and intraassay coefficients of variation for CRP were below 10%.