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.研究目的 本研究旨在对比围生期窒息伴或不伴缺氧缺血性脑病（hypoxic ischemic encephalopathy, HIE）新生儿出生时脐血（umbilical cord blood, UCB）中的神经元损伤与免疫炎症生物标志物，并以健康对照组为参照，同时评估上述标志物对HIE的预测能力。 研究设计 本研究为病例对照研究，纳入足月围生期窒息新生儿，于分娩时采集脐血并保存以用于批量检测。于出生后24小时采用临床Sarnat分期完成HIE诊断。本研究共纳入150例足月新生儿，其中50例HIE患儿、50例无HIE的围生期窒息（perinatal asphyxia, PA）患儿及50例健康对照者。采用超灵敏单分子阵列（Single molecule array, Simoa）检测法测定胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）、神经元生物标志物tau及神经丝轻链蛋白（neurofilament light protein, NFL）的浓度，并通过细胞因子筛选检测组合分析免疫炎症与感染相关标志物。 结果 相较于健康对照组，中重度HIE新生儿的GFAP、tau、NFL及多种细胞因子水平显著升高，且对HIE II/III型具有中等预测效能（曲线下面积AUC 0.681~0.827）。进一步分析显示，HIE II/III型患儿的GFAP、tau、白细胞介素6（Interleukin 6, IL-6）及白细胞介素8（Interleukin 8, IL-8）水平均高于PA/HIE I型患儿；IL-6水平在HIE II/III型与HIE I型患儿间亦存在显著差异。 结论 围生期窒息患儿脐血中的脑损伤与炎症生物标志物水平升高。相较于无HIE或HIE I型患儿，HIE II/III型患儿的多种生物标志物水平更高，提示上述标志物可辅助预测HIE II/III型。