Supplementary Material for: Neuro-specific and immuno-inflammatory biomarkers in umbilical cord blood in neonatal hypoxic-ischemic encephalopathy

Objectives To evaluate neuronal injury and immuno-inflammatory biomarkers in umbilical cord blood (UCB) at birth, in cases with perinatal asphyxia with or without hypoxic ischemic encephalopathy (HIE), compared with healthy controls and to assess their ability to predict HIE. Study design In this case-control study term infants with perinatal asphyxia were recruited at birth. UCB was stored at delivery for batch analysis. HIE was diagnosed by clinical Sarnat staging at 24 hours. Glial fibrillary acidic protein (GFAP), the neuronal biomarkers tau and neurofilament light protein (NFL), and a panel of cytokines were analysed in a total of 150 term neonates; 50 with HIE, 50 with asphyxia without HIE (PA) and 50 controls. GFAP, tau and NFL concentrations were measured using ultrasensitive Single molecule array (Simoa) assays, and a Cytokine Screening Panel was applied to analyse the immuno-inflammatory and infectious markers. Results GFAP, tau, NFL, and several cytokines were significantly higher in newborns with moderate and severe HIE compared to a control group and provided moderate prediction of HIE II/III (AUC 0.681-0.827). Furthermore, the levels of GFAP, tau, Interleukin 6 (IL-6) and Interleukin 8 (IL-8) were higher in HIE II/III cases compared with cases with PA/HIE I. IL-6 was also higher in HIE II/III compared with HIE I cases. Conclusions Biomarkers of brain injury and inflammation were increased in umbilical blood in cases with asphyxia. Several biomarkers were higher in HIE II/III vs. those with no HIE or HIE I, suggesting that they could assist in the prediction of HIE II/III.