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: 7.77). 09/2008; 54(10):1725-8. DOI: 10.1373/clinchem.2008.104711
Source: PubMed

ABSTRACT 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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    • "Five enzyme activities were measured using the modification of Li's multiplex assay by MS/MS as described by Zhang et al. [25]. Briefly, 5 assay cocktails were prepared from vials containing substrate and internal standard for each of five lysosomal enzymes: GAA, GLA, GBA, ASM, and GALC (Centers for Disease Control and Prevention, Atlanta, Georgia). "
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    • "j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m g m e [7] [8] [9] [10] [11] [12]. The quantification of enzyme activities in dried blood samples obtained at birth based on multiplex testing for a number of LSDs was suggested [13] [14]. However, benefits and ethical propriety of testing for some of the diseases included in the test panel have not yet been established [15]. "
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