摘要
目的:评估流动注射-串联质谱法(FIA-MS/MS)应用于溶酶体贮积症(LSDs)筛查的可行性。方法:病例对照研究。使用含酸性β葡糖脑苷脂酶(ABG)、酸性鞘磷脂酶(ASM)、β半乳糖脑苷脂酶(GALC)、α-L-艾杜糖苷酸酶(IDUA)、α半乳糖苷酶A(GLA)和酸性α葡糖苷酶(GAA)6种酶的底物及化学结构与相应产物类似的同位素内标准品缓冲液孵育,经液液萃取,氮气吹干,复溶后串联质谱分析酶反应产物和内标,计算酶活性。评价精密度、准确度、回收率、稳定性和携带污染。使用2022年7月至2023年3月上海市儿童医院2845份新生儿筛查样本和10例明确诊断的LSDs患儿样本进行临床验证。结果:6种低浓度酶活性检测值,批内变异系数(CV%)为4.650%~9.020%,批间CV%为6.990%~11.350%,高浓度酶活性检测值批内CV%为6.553%~8.690%,批间CV%为6.590%~12.850%;6个酶活性检测值与靶值的偏倚在-20.59%~20.45%;不同稀释浓度酶活性检测回收率为46.00%~136.67%,实测值与系列理论值间相关系数(R2)为0.986~0.997;稳定性评价6种酶活性差异均<15.00%;携带污染评价6种酶活性差异均<20.00%;6种酶活性在人群呈偏态分布,10例LSDs患儿酶活性明显低于切值,能有效检出相应疾病。结论:FIA-MS/MS检测ABG、ASM、GALC、IDUA、GLA和GAA有较高的精确性和准确度,有临床应用价值,可作为戈谢病、尼曼-皮克病A/B、克拉伯病、黏多糖贮积症Ⅰ型、法布里病和糖原贮积症Ⅱ型的筛查技术。
Objective To evaluate the feasibility of flow injection analysis-tandem mass spectrometry(FIA-MS/MS)for lysosomal storage diseases(LSDs)screening.Methods A case-control study.The enzyme substrates containing acidβ-glucocerebrosidase(ABG),acid sphingomyelinase(ASM),β-galactocerebrosidase(GALC),α-L-iduronidase(IDUA),α-galactosidase A(GLA)and acidα-glucosidase(GAA)were incubated with internal standard isotopes with similar chemical structure to the corresponding products.The enzyme reaction products and internal standards were analyzed by tandem mass spectrometry after liquid-liquid extraction and nitrogen blow-drying,and the enzyme activities were calculated.Precision,accuracy,recovery rate,stability and carrying pollution were evaluated.Clinical verification was performed using 2845 newborn screening samples and 10 confirmed diagnosis samples from Shanghai Children′s Hospital from July 2022 to March 2023.Results The activities of 6 low-concentration enzymes coefficient of variation(CV%)were 4.650%-9.020%in intraassay and 6.990%-11.350%in interassay,and high-concentration enzyme activities CV%were 6.553%-8.690%in intraassay and 6.590%-12.850%in interassay.The bias between the detected values and the target values of 6 enzyme activities was between-20.59%and 20.45%.The recovery rates of the enzyme activities at different dilution concentrations ranged from 46.00%to 136.67%,and the correlation coefficients(R2)between the measured values and the theoretical values were 0.986 to 0.997.The difference of 6 enzymes in stability was less than 15.00%.The difference of 6 enzymes in carrying pollution was less than 20.00%.The distribution of 6 enzyme activities was skewed in the population.The enzyme activities of 10 LSDs children were significantly lower than the cut-off value,which could effectively detect the corresponding diseases.Conclusions FIA-MS/MS has high precision and accuracy in the detection of ABG,ASM,GALC,IDUA,GLA and GAA,and is valuable in clinical application.It can be used as a screening technique for Gaucher disease,Niemann-Pick disease A/B,Krabbe disease,mucopolysaccharidosis typeⅠ,Fabry disease,and glycogen storage disease typeⅡ.
作者
王燕敏
田国力
纪伟
郭静
张潇分
周卓
Wang Yanmin;Tian Guoli;Ji Wei;Guo Jing;Zhang Xiaofen;Zhou Zhuo(Department of Neonatal Screening Center,Shanghai Children′s Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200040,China)
出处
《中华实用儿科临床杂志》
CAS
CSCD
北大核心
2024年第5期327-331,共5页
Chinese Journal of Applied Clinical Pediatrics
基金
上海市重中之重临床重点专科建设项目(2017ZZ02019)
上海市科委项目(22Y11906900)
上海交通大学“交大之星”计划医工交叉研究项目(YG2022QN097)
上海市儿童医院国家自然科学基金培育专项(2021YGZQ09)。
关键词
溶酶体贮积症
流动注射-串联质谱
酶活性
新生儿筛查
Lysosome storage disease
Flow injection analysis-tandem mass spectrometry
Enzyme activity
Neonatal screening
