Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry
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.
Full-textDOI: · Available from: Carole Elbin, Jan 16, 2015
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Article: Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry
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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. . Briefly, substrate and internal standard cocktails for each lysosomal enzyme were combined with DBS extract and incubated overnight. "
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.06/2015; 3. DOI:10.1016/j.ymgmr.2015.04.001
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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. . 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). "
ABSTRACT: Fluorometric and tandem mass spectrometry assays can be used to measure lysosomal enzyme activities in dried blood spots (DBS). The effect of DBS preparation, storage and shipping was evaluated on the activities of acid α-glucosidase, acid α-galactosidase, acid β-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase. Whole blood from normal donors was used to prepare DBS following Clinical and Laboratory Standards Institute guidelines and by several deviations. Some DBS were subjected to various treatments, storage and shipping conditions. The activity of 5 lysosomal enzymes (GAA, GLA, GBA, ASM, and GALC) was measured using tandem mass spectrometric and fluorometric (GAA only) assays with 2 distinct and commonly used synthetic substrates. Enzyme activities were strongly affected by the way DBS were prepared and stored. Exposure of DBS to elevated heat and humidity can destroy enzyme functions rapidly. DBS prepared from poorly mixed blood caused significant variation on enzyme activities. EDTA, but not heparin, as an anti-coagulant gave more precise results. The study confirmed the importance of proper and consistent DBS preparation and storage when screening for deficiencies of lysosomal enzymes.Clinica chimica acta; international journal of clinical chemistry 03/2011; 412(13-14):1207-12. DOI:10.1016/j.cca.2011.03.012 · 2.76 Impact Factor
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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      . The quantification of enzyme activities in dried blood samples obtained at birth based on multiplex testing for a number of LSDs was suggested  . However, benefits and ethical propriety of testing for some of the diseases included in the test panel have not yet been established . "
ABSTRACT: Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies, resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma, as well as in various tissues. We devised and validated a straightforward, but accurate and precise tandem mass spectrometry methodology coupled to high performance liquid chromatography (LC-MS/MS) for the quantification of GAGs in urine. The method is applicable to the investigation of patients with MPS I, II, and VI, by quantifying dermatan sulfate (DS) and heparan sulfate (HS) in urine. We analyzed urine samples from 28 MPS patients, aged 1 to 42 years, and 55 control subjects (41 days to 18 years old). Levels of DS and HS in urine from healthy controls of all ages were below the limit of quantification. The levels of DS and HS in urine from 6 treated patients with MPS I were lower than in 6 untreated patients in DS (0.7-45 vs 9.3-177 mg/mmol creat) and HS (0-123 mg/mmol creatinine vs 38-418 mg/mmol creatinine); similar results were obtained for 9 patients with MPS II and 7 patients with MPS VI. Analyses were performed on as little as 250 μL of urine. Methanolysis took 75 min per sample; the total analysis run time for each LC-MS/MS injection was 8 min. Results indicate that the method is applicable to a wide variety of situations in which high accuracy and precision are required, including the evaluation of the effectiveness of existing and emerging treatments.Molecular Genetics and Metabolism 09/2010; 102(1):49-56. DOI:10.1016/j.ymgme.2010.09.003 · 2.83 Impact Factor