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：本研究旨在对比围产期窒息伴或不伴缺氧缺血性脑病（hypoxic ischemic encephalopathy, HIE）患儿出生时的脐血（umbilical cord blood, UCB）中的神经元损伤与免疫炎症生物标志物水平，并与健康对照人群进行比较，同时评估上述标志物预测HIE的效能。
研究设计：本研究为病例对照研究，纳入出生时招募的足月围产期窒息患儿。脐血于分娩时留存，用于批量检测分析。缺氧缺血性脑病于出生后24小时通过临床Sarnat分期进行诊断。本研究共纳入150名足月新生儿，其中50名HIE患儿、50名围产期窒息无HIE患儿（PA）以及50名健康对照者，对胶质纤维酸性蛋白（GFAP）、神经元生物标志物tau、神经丝轻链蛋白（neurofilament light protein, NFL）以及一组细胞因子进行了检测。GFAP、tau及NFL的浓度采用超灵敏单分子阵列（Single molecule array, Simoa）技术进行检测，同时使用细胞因子筛查试剂盒分析免疫炎症与感染相关标志物。
研究结果：与健康对照组相比，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级具有中等预测效能（曲线下面积AUC为0.681~0.827）。此外，与PA或HIE I级患儿相比，HIE II/III级患儿的GFAP、tau、白细胞介素6（Interleukin 6, IL-6）及白细胞介素8（Interleukin 8, IL-8）水平更高；且HIE II/III级患儿的IL-6水平也高于HIE I级患儿。
研究结论：围产期窒息患儿的脐血中脑损伤与炎症相关生物标志物水平升高。多项生物标志物在HIE II/III级患儿中的表达水平高于无HIE患儿及HIE I级患儿，提示上述标志物可辅助预测HIE II/III级病变。