The artificial-liver blood-purification system can effectively improve hypercytokinemia for COVID-19

Since December 2019, the outbreak of Coronary Virus Disease 2019 (COVID-19) occurred in Wuhan, and rapidly spread across the country, and the world. As the number of diagnoses and deaths continues to rise, this has become the focus of international public health. COVID-19 is highly contagious, and there is no effective treatment. New treatment strategies are urgently needed to improve the treatment success rate of severe and critically ill patients. Increasing evidence has shown that cytokine storm plays an important role in the progression of COVID-19. The artificial liver blood purification system (ALS) is expected to improve the cytokine storm. In the present study, the levels of cytokines and chemokines were detected in 12 COVID-19 patients before and after ALS, and exciting results were obtained. The present study has directly proven that ALS can block the cytokine storm, rapidly remove the inflammatory mediators, and hopefully suppress the progression of the disease, providing a new idea for the clinical treatment of COVID-19.

Methods
The present study included 12 critically ill patients with COVID-19, who received treatment with the ALS from January 15, 2020 to March 31, 2020. Each patient’s gender, age, symptoms, complications and disease severity were recorded in detail. A written informed consent was obtained from each patient, or their legally authorized representative. The present study was approved by the Institutional Review Board of the First Affiliated Hospital, School of Medicine, Zhejiang University .

Each patient received three treatment courses with the ALS, and peripheral blood was collected before and right after each treatment course (T1B, T1A, T2B, T2A, T3B and T3A). The EDTA anticoagulated blood was centrifuged at 3,000 rpm for five minutes, and stored at −80°C until assayed.

The magnetic bead-based multiplex immunoassays were developed using the Bio-Plex ProTM Human 48-plex Cytokine Screening Panel, according to manufacturer’s instructions, using the Bio-Plex 200 suspension array system (Bio-Rad, Hercules, CA, USA) in a BSL-2 laboratory. The primary data were analyzed using the Bio-Plex Manager Software, version 6.1.1.

Statistical analysis

The variables were expressed in medians for the statistical analysis. The paired serum cytokine/chemokine results generated at different time points for the same patient were analyzed using the Wilcoxon signed rank test. The statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Statistical analyses of the data were performed using the SPSS software.

自2019年12月起，新型冠状病毒肺炎（Coronary Virus Disease 2019，COVID-19）疫情率先在武汉暴发，并迅速蔓延至全国乃至全球。随着确诊病例与死亡人数持续攀升，该疫情已成为国际公共卫生领域的关注焦点。COVID-19传染性极强，目前尚无特效治疗方案，亟需探索全新治疗策略以提升重症及危重症患者的救治成功率。越来越多的研究证据表明，细胞因子风暴在COVID-19的病情进展中发挥关键作用。人工肝血液净化系统（artificial liver blood purification system, ALS）有望改善细胞因子风暴状态。本研究对12例COVID-19患者接受ALS治疗前后的细胞因子与趋化因子水平进行了检测，并取得了令人振奋的研究结果。本研究直接证实了ALS可阻断细胞因子风暴、快速清除炎症介质，或可抑制病情进展，为COVID-19的临床治疗提供了全新思路。

方法
本研究纳入2020年1月15日至2020年3月31日期间接受ALS治疗的12例COVID-19危重症患者。研究人员详细记录了每位患者的性别、年龄、临床症状、并发症及疾病严重程度。所有患者或其法定授权代表均签署了书面知情同意书，本研究已通过浙江大学医学院附属第一医院伦理审查委员会审批。

每位患者接受3个疗程的ALS治疗，并在每个疗程开始前及结束后即刻采集外周血样本（分别标记为T1B、T1A、T2B、T2A、T3B及T3A）。采集的乙二胺四乙酸（Ethylene Diamine Tetraacetic Acid, EDTA）抗凝血以3000转/分钟离心5分钟，随后置于-80℃冰箱保存以待后续检测。

本研究采用基于磁珠的多重免疫检测方法，依照制造商说明书，使用Bio-Plex 200悬浮阵列系统（美国加利福尼亚州赫拉克勒斯市伯乐（Bio-Rad）公司产品），在生物安全二级（BSL-2）实验室中完成检测，检测试剂盒为Bio-Plex Pro™人类48因子细胞因子筛选面板。原始数据通过Bio-Plex Manager软件6.1.1版本进行分析处理。

统计学分析
统计学分析中，计量资料以中位数表示。针对同一患者不同时间点获取的配对血清细胞因子/趋化因子检测结果，采用Wilcoxon符号秩检验（Wilcoxon signed rank test）进行分析。本研究的统计分析分别通过GraphPad Prism 5软件（美国加利福尼亚州圣地亚哥市GraphPad软件公司）及SPSS软件完成。