脓毒症患者中性粒细胞胞外诱捕网的定量研究

Quantitative analysis of neutrophil extracellular traps in patients with sepsis

目的建立流式细胞术(FCM)检测中性粒细胞胞外诱捕网(NETs)的表达方法,探讨NETs在脓毒症相关凝血功能障碍(SAC)患者中的临床诊断价值。方法收集2018年5月至2019年6月苏州大学附属第二医院50例非脓毒症者和21例脓毒症患者抗凝血。脓毒症患者符合3.0定义的诊断标准,血培养鉴定出革兰氏阴性菌,D二聚体含量>0.5 mg/L,彩色多普勒超声检查显示血栓形成,即时收集外周血样。采用别藻蓝蛋白(APC)标记的白细胞分化抗原66b(CD66b)识别多型分叶核细胞(PMNs),异硫氰酸荧光素(FITC)标记的抗髓过氧化物酶(MPO)抗体耦联PMNs膜表面MPO,Sytox orange(SO)核酸染料标记脱氧核糖核酸(DNA),建立FCM检测全血NETosis相关标志物方法。7-氨基放线菌素D(7-AAD)染料观察溶血对细胞膜完整性的影响,进行NETosis相关标志物(SO/MPO)的特异性检测。受试者工作特征(ROC)曲线分析NETosis相关标志物和白细胞总数(WBC)在SAC患者中的灵敏度和特异性,用Mann-Witney统计量的非参数法估计ROC曲线下面积(AUC),AUC的比较采用配对设计的两相关诊断试验差异假设检验。结果全血分离的PMNs与FACS试剂溶血后的PMNs对7-AAD的阴性表达率比较,两者差异无统计学意义[(95.1±6.6)%比(93.5±5.7)%,t=1.984,P>0.05]。NETosis相关标记物SO/MPO不能识别Etoposide药物诱导的PMNs凋亡(Apoptosis)。ROC曲线分析SAC患者的AUC,NETosis相关标记物取2.5%时的AUC高于WBC取8.0×109/L时的AUC(0.97比0.72,Z=2.011,P<0.05),差异有统计学意义。结论 FCM检测外周全血PMNs上NETosis相关标志物具有良好的敏感性、特异性,在SAC诊断中有重要临床价值。

Objective To establish the method based on flow cytometry for detecting the expression of neutrophil extracellular traps(NETs),and to discuss the clinical diagnostic value of NETs in patients with sepsis-associated coagulopathy(SAC).Methods 21 blood samples of sepsis patients and 50 non-sepsis patients were collected from May 2018 to June 2019 in the Second Affiliated Hospital of Soochow University.Sepsis patients were defined by the diagnostic criteria of sepsis 3.0.Gram-negative bacteria were identified by blood culture,D-dimer content of>0.5 mg/L,color Doppler ultrasound showed thrombosis,and peripheral blood samples were collected immediately.Cluster of Differentiation 66b(CD66b)labeled by Allophycocyanin(APC)was used to identify polymorphonuclear cells(PMNs),and Anti myeloperoxidase(anti-MPO)antibody labeled by fluorescein isothiocyanate(FITC)was coupled with membrane surface MPO,and deoxyribonucleic acid(DNA)was labeled by the nucleic acid staining agent Sytox orange(SO).Then flow cytometry was employed to detect the related markers of NETosis in whole blood.7-aminoactinomycin D(7-AAD)dye was used to observe the effect of hemolysis on cell membrane integrity.The specificity of NETosis related markers(SO/MPO)were evaluated.Receiver operating characteristic(ROC)curve was drawn to analyze the sensitivity and specificity of NETosis related markers and total white blood cell count(WBC)in patients with SAC.The area under ROC curve(AUC)was calculated by nonparametric method of Mann-Witney statistics.The significance test of paired design was used to compare AUC.Results The negative expression rate of 7-AAD was compared between PMNs isolated from whole blood and PMNs hemolyzed by FACS Lysing Solution.There was no significant difference between these two groups[(95.1±6.6)%vs.(93.5±5.7)%,t=1.984,P>0.05].SO/MPO,the NETosis related markers,could not recognize the neutrophil apoptosis induced by Etoposide.The AUC of patients with SAC was analyzed by ROC curve.The AUC at 2.5%of NETosis related markers was higher than that at 8.0×109/L of WBC(0.97 vs.0.72,Z=2.011,P<0.05),the difference was statistically significant.Conclusion The method of detection of NETosis related markers in peripheral blood PMNs based on flow cytometry technology has good sensitivity and specificity,which has important potential clinical value in the diagnosis of SAC.