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

Genzyme Corporation, Framingham, MA 01701, USA.
Clinical Chemistry (Impact Factor: 7.91). 09/2008; 54(10):1725-8. DOI: 10.1373/clinchem.2008.104711
Source: PubMed


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.
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.
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.
The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.

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Available from: Carole Elbin, Jan 16, 2015
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    • "The amount of product formed during the reactions was quantified by calculating the ratio of ion abundance of the product formed (P) versus the internal standard (P/IS) from backgroundsubtracted data. For the multiplex assay, the enzyme reaction method and LC/MS/MS analysis were performed as described in Zhang et al. [8]. Briefly, substrate and internal standard cocktails for each lysosomal enzyme were combined with DBS extract and incubated overnight. "
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    Molecular Genetics and Metabolism Reports 06/2015; 3. DOI:10.1016/j.ymgmr.2015.04.001
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    • "Many screening programs use a single-stage multi-analyte approach, where multiple markers of multiple diseases can be screened in a single analysis [5]. Mass spectrometry-based screening assays from DBS are now available for PKU, MCAD [6], maple syrup urine disease [7] , homocystinurea [8], and lyososomal storage disorders [9]. "
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    ABSTRACT: Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs. Figure ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-013-0658-1) contains supplementary material, which is available to authorized users.
    Journal of the American Society for Mass Spectrometry 06/2013; 24(8). DOI:10.1007/s13361-013-0658-1 · 2.95 Impact Factor
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    • "Since the MS/MS techniques were developed for newborn screening of LSDs, several methods for multiplex MS/MS have been introduced; however, the maximum number of diseases reported to be diagnosed by using multiplex MS/MS is 5 [7, 8]. To our knowledge, this is the first study that reports the feasibility of multiplex MS/MS for diagnosing 6 LSDs, including MPS I, in newborns. "
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    ABSTRACT: We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome. We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods. The intra- and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns. The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.
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