Direct Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening

Abstract

Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. Methods: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid ␣-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approximately one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated,

Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders

“…According to our measurements (Li et al 2004b), the Pompe enzyme constitutes only about 30% of total acid α-glucosidase activity in a detergent extract from human neutrophils. Thus, purified lymphocytes, fibroblasts, or muscle tissue have been recommended for the biochemical diagnosis of Pompe disease (Koster et al 1974;Shin et al 1985).…”

“…The product is the 6-carbon fatty acyl ceramide, and the internal standard is the ceramide with a 4-carbon fatty acyl chain. Since all products and internal standards have different masses, the Krabbe, Gaucher and Niemann-Pick-A/B enzymes can be analysed together by a single infusion into the tandem mass spectrometer (Li et al 2004b). All substrates and internal standards are available from Avanti Polar Lipids, Inc.…”

“…We also developed a tandem MS assay for analysis of Fabry disease, caused by the deficiency of the enzyme acid α-galactosidase A (Li et al 2004b). The assay is illustrated in Fig.…”

Selective Screening for Lysosomal Storage Diseases with Dried Blood Spots Collected on Filter Paper in 4,700 High-Risk Colombian Subjects

“…This finding was demonstrated in the present study, where total catalytic activity was not different between the healthy controls and the patients (p < 0.176). A modified technique was used based on the one reported by Kallwass et al (2007) and Li et al (2004), which permitted the discrimination of the activity of the different isoforms using acarbose as an inhibitor. This method achieved significant differences between the controls and the individuals affected by Pompe disease (see Table 3).…”

“…The analysis of a-glucosidase was performed according to a standardized methodology described by Kallwass et al (2007) and Li et al (2004). The analysis of arylsulfatase B was performed using a standardized methodology based on the analytical principle of the leukocyte analysis proposed by Shapira et al (1989) and Civallero et al (2006).…”