串联质谱技术在X-连锁肾上腺脑白质营养不良病筛查中的应用价值  

Role of tandem mass spectrometry for screening X-linked adrenoleukodystrophy

作　　者：王燕敏[1] 田国力[1] 纪伟 WANG Yanmin;TIAN Guoli;JI Wei(Neonatal Screening Center,Children's Hospital of Shanghai,Children's Hospital of Shanghai Jiaotong University,Shanghai 200040,China)

机构地区：[1]上海市儿童医院上海交通大学附属儿童医院新生儿筛查中心

出　　处：《检验医学》2019年第12期1059-1065,共7页Laboratory Medicine

基　　金：上海市科学技术委员会科研项目（18441905100）;上海市重中之重临床重点专科建设项目（2017ZZ02019）;上海市第四轮公共卫生三年行动计划重点学科建设项目（15GWZK0401）;上海交通大学多学科交叉项目（YG2015QN27）

摘　　要：目的建立串联质谱(MS/MS)检测极长链酰基肉碱(VLCAC)和溶血磷脂酰胆碱(LPC)的方法,初步探讨VLCAC和LPC对X-连锁肾上腺脑白质营养不良病(X-ALD)的诊断价值。方法选取被明确诊断为X-ALD的患儿10例和行遗传代谢性疾病筛查的正常新生儿3530名。收集所有对象的滤纸干血片样本,使用含稳定同位素内标[2H3-二十六碳酰基肉碱(2H3-C26)和2H4-二十六碳溶血磷脂酰胆碱(2H4-C26:0-LPC)]的溶剂萃取滤纸干血片样本中的VLCAC和LPC(非衍生化法),直接采用MS/MS检测二十碳酰基肉碱(C20)、二十二碳酰基肉碱(C22)、二十四碳酰基肉碱(C24)、二十六碳酰基肉碱(C26)、二十碳溶血磷脂酰胆碱(C20:0-LPC)、二十二碳溶血磷脂酰胆碱(C22:0-LPC)、二十四碳溶血磷脂酰胆碱(C24:0-LPC)和二十六碳溶血磷脂酰胆碱(C26:0-LPC)。对建立的MS/MS方法进行方法学评价(精密度、准确度、线性)。采用偏最小二乘法分析各项指标对疾病的贡献度。采用受试者工作特征(ROC)曲线评估各项指标诊断X-ALD的效能。结果低水平C26和C26:0-LPC的批内变异系数(CV)分别为4.16%和7.75%,批间CV分别为8.29%和9.17%;高水平C26和C26:0-LPC的批内CV分别为5.57%和8.68%,批间CV分别为7.45%和8.11%。准确度为93.25%~104.94%。C26和C26:0-LPC实测值与预测值之间的决定系数(r2)分别为0.996和0.994。除C20、C24:0-LPC/C20:0-LPC、C24:0-LPC/C22:0-LPC、C26:0-LPC/C20:0-LPC外,其他指标正常对照组与X-ALD组差异均有统计学意义(P<0.05)。C26、C26/C22比值和C26:0-LPC对疾病的贡献度最高(偏回归系数分别为0.1753、0.1300和0.0792)。ROC曲线分析结果显示,C26和C26/C22比值诊断X-ALD的敏感性均为100%,特异性均为100%,曲线下面积(AUC)均为1.0;C26:0-LPC诊断X-ALD的敏感性为100%,特异性为72.9%,AUC为0.972。结论非衍生化MS/MS检测VLCAC和LPC有较高的精密度和准确度,C26或可作为诊断X-ALD的生物标志物。Objective To establish a tandem mass spectrometry(MS/MS)for the determinations of very long chain acylcarnitine(VLCAC)and lysophosphatidylcholine(LPC),and to evaluate the diagnostic efficiency of VLCAC and LPC in the diagnosis of X-linked adrenoleukodystrophy(X-ALD).Methods A total of 3530 samples of dried blood spots for screening genetic metabolic diseases and 10 samples of dried blood spots from children with X-ALD were collected.VLCAC and LPC were extracted from dried blood spots by solvent extraction with standard substances containing stable isotopic markers of 2H3-hexacosanoylcarnitine(2H3-C26)and 2H4-C26:0 lysophosphatidylcholine(2H4-C26:0-LPC)direct injection in tandem mass spectrometer.Eicosanoylcarnitine(C20),docosanoylcarnitine(C22),tetracosanoylcarnitine(C24),hexacosanoylcarnitine(C26),C20:0 lysophosphatidylcholine(C20:0-LPC),C22:0 lysophosphatidylcholine(C22:0-LPC),C24:0 lysophosphatidylcholine(C24:0-LPC)and C26:0 lysophosphatidylcholine(C26:0-LPC)were detected.Methodological evaluation(precision,accuracy and linearity)of the established MS/MS was performed.Partial least square method was used to analyze the contribution of each index to disease.Receiver operating characteristic(ROC)curve was used to evaluate the efficiency of each index for the diagnosis of X-ALD.Results The withinrun coefficients of variation(CV)of low-concentration C26 and C26:0-LPC were 4.16% and 7.75%,and the between-run CV were 8.29% and 9.17%,respectively.The within-run CV of high-concentration C26 and C26:0-LPC were 5.57 %and 8.68%,and the between-run CV were 7.45% and 8.11%,respectively.The accuracies were 93.25%-104.94%.The determination coefficients(r2)between the measured values of different dilution concentrations and the theoretical concentration were 0.996 and 0.994.Except for C20,C24:0-LPC/C20:0-LPC,C24:0-LPC/C22:0-LPC and C26:0-LPC/C20:0-LPC,the difference between control group and X-ALD group was statistically significant(P<0.05).C26,C26/C22 ratio and C26:0-LPC had the highest contribution to disease(the partial re

关 键 词：极长链酰基肉碱 溶血磷脂酰胆碱 串联质谱 X-连锁肾上腺脑白质营养不良病 

分 类 号：R446.1[医药卫生—诊断学]