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.


Available from: Carole Elbin, Jan 16, 2015
    • "The pathological application of dehydrated blood stain specimen on blotter paper for the quantification of chemical constituents has emerged as a novel minimally invasive option for the screening of disease from blood samples, which can be used for affordable diagnosis of patients' disease. Analysis of dried blood stains has been known to doctors [1, 2], and medical researchers are using this technique for screening of various diseases cautiously after experimental studies and clinical trials on this method345678910111213. Once validation is proved with experimental trials and clinical tests, this disease screening method using dried blood stain pattern analysis can provide a cost-effective affordable healthcare tool for rapid, and/or instant pathological screening of disease from a patient's blood sample. "
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    • "THE AMERICAN BIOLOGY TEACHER VOLUME. 77, NO. 6, AUGUST 2015 446 tool (Zhang et al., 2008 "
    [Show abstract] [Hide abstract] ABSTRACT: Using Pompe disease as a context affords the opportunity for students to consider multiple biological concepts and embraces the Next Generation Science Standards Disciplinary Core Ideas Structure and Function (LS1.A) and Inheritance of Traits (LS3.A) as well as Crosscutting Concepts Structure and Function and Cause and Effect. These crosscutting concepts are very much interrelated as we consider progression of disease from the molecular to the organismal level. The concepts are repeatedly emphasized, providing explicit instructional support for students to develop a cumulative, coherent, and usable understanding of science and engineering. DNA, proteins, enzymes, genetics, and human disease are taught together through the story of patients with Pompe disease as students engage in a simulated clinical assay and genetic analysis and present their findings in grand rounds. The activity is one of multiple lessons sequenced to scaffold student understanding of clinical and translational science, starting with a first-person perspective of a father who loses his infant son to Pompe and concluding with a role play based on actual events surrounding approval of human clinical trials of gene therapy for Pompe disease. © 2015 by National Association of Biology Teachers. All rights reserved.
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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. "
    [Show abstract] [Hide abstract] ABSTRACT: Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls.
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