New Strategy for the Screening of Lysosomal Storage Disorders: The Use of the Online Trapping-and-Cleanup Liquid Chromatography/Mass Spectrometry

Abstract

The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by "Pompe", 6 by "Gaucher", 12 by "Fabry", 3 by "Niemann-Pick" A/B, and 2 by "Krabbe" diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.

Newborn Screening for Lysosomal Storage Disorders in Hungary

“…An experienced operator can perform the cleanup of 4,500 samples, the daily workload of an average size screening laboratory, within 4-6 h. Recent introduction of online HPLC sample cleanup technology would save the time-consuming cleanup procedure as well as consumables but requires longer analysis time (LaMarca et al 2009) or more sophisticated equipment (Kasper et al 2010). However, using online HPLC cleanup on MS/MS, the instrument very likely can be used only for LSD screening and is blocked for other analysis.…”

Simplified Newborn Screening Protocol for Lysosomal Storage Disorders

“…The first such protocol was described in a report by la Marca et al (24 ), in which they suggested that online multidimensional HPLC sample preparation eliminated the need for laborious preanalytical steps with the use of a homebuilt multidimensional LC method in which a POROS cleanup column (Applied Biosystems) and an analytical C18 column were used.…”

Newborn screening for Krabbe disease in New York State: the first eight years’ experience

“…A multiplex tandem mass spectrometry (MS/MS) assay for five LSDs (KD, Gaucher, Pompe, Niemann-Pick, and Fabry diseases) was developed 13 and optimized for use on dried blood spots. 15,18,19 There are challenges to adding LSDs to NBS panels, including the need for expensive instrumentation and adequate quality control materials (especially dried blood spots from infants with KD), 14,20 diagnostic laboratories for confirmatory testing with short turnaround times, infrastructure for rapid referral and urgent transplantation in newborns with EIKD, protocols for follow-up, and clinicians experienced in interpreting and relaying the full spectrum of NBS results, including variability in enzyme activity and identification of sequence variants of unknown significance or variable expressivity. 21 Early studies showed that a small cohort of infantile KD patients who received umbilical cord blood transplantation from unrelated donors prior to symptom onset had preserved neurological function.…”