Newborn Screening, Biochemistry and Pharmacology Laboratory, Pediatric Neurology, Unit and Laboratories, Meyer Children's University Hospital, Florence, Italy.
Analytical & Measuring Instruments Division Shimadzu Corporation, Kyoto, Japan.
J Pharm Biomed Anal. 2019 Feb 20;165:135-140. doi: 10.1016/j.jpba.2018.12.002. Epub 2018 Dec 3.
New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.
新型治疗方法和更优的溶酶体贮积症（LSD）诊断策略在干血斑（DBS）上的应用，催生了若干试点新生儿筛查项目。我们在先前发表的方案基础上，设计了一种新的 6 重分析检测，该检测基于单次 DBS 点刺，孵育于缓冲液中，缓冲液中包含了一组酶底物（GAA、GLA、ASM、GALC、ABG 和 IDUA）。新方案中采用了新的捕获和净化程序，利用与分析柱在线连接的灌注色谱，对经酶促反应后的样品进行分析物分离。4.5 分钟后即可得到结果。我们测试了几种孵育时间，以减少样品制备时间，并提高分析准确性和重复性，同时也确保在产物线性积累的时间窗口内对反应进行淬灭。所收集的数据表明，孵育 4 小时即可达到良好的反应效率，而不会对灵敏度产生任何影响。该方法对于不同的仪器配置均具有通用性和稳健性。快速的样品制备和运行时间允许实现高通量的样本处理；这在新生儿筛查程序中是一个优势。该方法也可用于诊断目的，可在数小时内快速诊断。
