Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry

Abstract

Background: Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories. Methods: We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with a lysosomal storage disorder (LSD) were tested using the modified assay. Results: In our study, the median enzyme activity measured in adults was generally increased 2–3–fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples. Conclusions: The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.

Newborn Screening for Lysosomal Storage Disorders in Hungary

“…The vials contained substrate and internal standard in a molar ratio of about 50:1. Reagents were obtained lyophilized and dissolved in appropriate buffers as previously described (Zhang et al 2008). Chemicals for preparation of buffers and inhibitor solutions were obtained from VWR International and Sigma-Aldrich, respectively.…”

“…Sample preparation was performed as previously described (Li et al 2004;Zhang et al 2008). Briefly, 3.2 mm spots from the DBS and quality control material (CDC, Atlanta, USA) were punched into 96-well polypropylene plates (Greiner Bio-One) using a Wallac DBS Puncher (Perkin Elmer, Massachussets, USA).…”

“…The definition of the cutoffs was based on the experience that affected patients have virtually no enzyme activity (Zhang et al 2008), but they were also kept low in order to keep the potential recall rate low. Only in case of Fabry disease the cutoffs used were slightly above the 0.5th percentile.…”

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.…”

Mass spectrometry in clinical chemistry: the case of newborn screening

“…Some methods were developed for the detection of lysosomal storage disorders (LSDs) and even if for some of them a reliable and relatively simple test is available, a reasonable associated therapy could be still not available [29][30][31][32][33][34][35][36][37][38]. It is not the task of this writer to produce a statement that the LSDs should be included in newborn screening panels, but some of these disorders seem to meet the criteria for inclusion, including basal availability of a simple test, a combination therapy that modifies G. la Marca / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx-xxx the natural course of the disease, early diagnosis that allows genetic counseling and prenatal diagnosis in families with an affected baby.…”