Pickens C Austin, Courtney Elya, Isenberg Samantha L, Cuthbert Carla, Petritis Konstantinos
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
Clin Chem. 2023 Apr 28;69(5):470-481. doi: 10.1093/clinchem/hvad007.
Classical homocystinuria (HCU) results from deficient cystathionine β-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS.
Dried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS).
Interferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent-free quantification of tHcy by FIA-MS/MS in first-tier NBS. The combination of tris(2-carboxyethyl)phosphine (TCEP) and NEM yielded significantly less matrix effects compared to dithiothreitol (DTT) and NEM. Analysis of clinical specimens demonstrated that the method could distinguish between HCU-positive, presumptive normal newborns, and newborns receiving total parenteral nutrition.
Here we present the first known validated method capable of screening tHcy in DBS during FIA-MS/S first-tier NBS.
经典型同型胱氨酸尿症（HCU）是由胱硫醚β-合酶活性缺乏引起的，导致蛋氨酸（Met）和同型半胱氨酸（Hcy）水平升高。新生儿筛查（NBS）旨在通过评估一级筛查中的Met浓度来识别无症状新生儿中的HCU。然而，与Hcy不同，Met检测会导致大量假阳性和假阴性结果。因此，在一级筛查中直接检测Hcy将是用于NBS的更好生物标志物。
使用干血斑（DBS）质量控制和剩余临床标本进行分析。研究了几种还原试剂和马来酰亚胺试剂，以辅助总Hcy（tHcy）的定量。开发并验证的检测方法通过流动注射分析-串联质谱（FIA-MS/MS）进行。
确定了tHcy测量的干扰物，因此采用了Hcy的选择性衍生化。使用N-乙基马来酰亚胺（NEM）对Hcy进行选择性衍生化，可通过FIA-MS/MS在一级NBS中对tHcy进行无干扰定量。与二硫苏糖醇（DTT）和NEM相比，三（2-羧乙基）膦（TCEP）和NEM的组合产生的基质效应明显更小。临床标本分析表明，该方法可以区分HCU阳性、疑似正常新生儿和接受全胃肠外营养的新生儿。
在此，我们展示了第一种已知的经过验证的方法，该方法能够在FIA-MS/S一级NBS期间对DBS中的tHcy进行筛查。
