A tandem mass spectrometry triplex assay for the detection of Fabry, Pompe, and mucopolysaccharidosis-I (Hurler)

Abstract

Background: We sought to develop a tandem mass spectrometry assay in which the enzymatic activities of 3 lysosomal enzymes (α-glucosidase, α-galactosidase A, and α-l-iduronidase) could be quantified in dried blood spots by using a single assay buffer.

Methods: A 3-mm dried blood spot punch was incubated in a single assay buffer with 3 different substrates and internal standards. The sample was processed by a simple liquid-liquid extraction by using ethyl acetate. The extract was dried down and resuspended in solvent for injection into the tandem mass spectrometer. Products and internal standards were monitored by multiple reaction monitoring.

Results: Assay for the 3 lysosomal enzymes was successfully achieved with acceptable statistics. The assay can be performed by using a minimal quantity of disposable supplies and equipment. The entire procedure fits into a 48-h cycle including data analysis. Data from 5990 anonymous newborn dried blood spots showed an approximate bell-shaped distribution of enzymatic activities (mean values of 19.0, 11.5, and 3.5 μmol · h(-1) · (L blood)(-1) for α-glucosidase, α-galactosidase A, and α-l-iduronidase, respectively. Blank values obtained in the absence of blood were 0.13, 0.24, and 0.45 μmol · h(-1) · (L blood)(-1), respectively). By assaying 3 enzymes at once, problematic samples are spotted for reanalysis if enzyme activity values are low for all enzymes (for example, if insufficient blood is present in the assay).

Conclusions: This method demonstrates that a triplex assay in a single buffer and with minimal supplies and labor can be adapted to a high-throughput newborn screening laboratory for the analysis of Pompe, Fabry, and mucopolysaccharidosis-I (Hurler) diseases.

Fig. 1. Enzyme activity versus pH and buffer components

Assays were performed with DBSs from a healthy adult. Data are plotted as the mean and SD of triplicate assays carried out as described in the online Data Supplement.

Fig. 2. Typical work flow of the assay run in a newborn screening laboratory

Assay results are obtained within 48 h of the beginning of the procedure.

Fig. 3. Activity distribution of GLA, GAA, and IDUA from 5990 anonymous newborn blood spots

Assays were performed in the Washington State newborn screening laboratory by using the triplex assay.