Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening
Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening
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DOI：
10.1373/clinchem.2004.035907
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发表时间：
2004-10-01
影响因子：
9.3
通讯作者：
Gelb, MH
中科院分区：
文献类型：
--
作者：
Li, YJ;Scott, CR;Gelb, MH
背景：新生儿筛查导致Fabry， Gaucher, Krabbe， Niemann-Pick A/B和Pompe病的溶酶体酶缺乏是有必要的，因为这些综合征的治疗现在是可用的或预计在近期。我们描述了一种对所有五种溶酶体酶的多重筛选方法，该方法使用含有干血斑的新boms筛选卡作为酶源。方法：采用底物盒和内标法直接定量酶活性，串联质谱法检测酶产物。再水化干血斑与酶底物孵育。采用液液萃取法，硅胶固相萃取法去除缓冲成分。阿卡波糖作为中性粒细胞中存在的一种干扰性酸性-葡萄糖苷酶的抑制剂，可以选择性地分析与庞贝病有关的溶酶体酶。结果：我们分析了5例戈谢病、5例尼曼-匹克A/B病、11例Pompe病、5例Fabry病和12例Krabbe病的干血斑，在所有病例中，酶活性均低于杂合携带者和健康非携带者收集的最低活性。在5-9个杂合携带者中测量的酶活性约为15-32个健康个体的一半，但携带者和健康个体的数据集之间存在部分重叠。结论：对5种疾病均检测到感染个体。该分析可以很容易地实现自动化，并且预期的试剂和供应成本完全在新轰炸机筛选中心的预算限制之内。(C) 2004美国临床化学学会。
Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newbom-screening cards containing dried blood spots as the enzyme source.Methods: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated With the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid a-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed.Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approximately one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals.Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newbom-screening centers. (C) 2004 American Association for Clinical Chemistry.