Objective To assess the performance of a tandem mass spectrometry technology in a newborn screening laboratory to simultaneously measure α-galactosidase, acid-α-glucosidase, and α-L-iduronidase for the detection of infants at risk to develop Fabry, Pompe, or Mucopolysaccharidosis-I (MPS-I) diseases. Study design Enzyme activity was assayed from a 3.2-mm punch from 100,000+ anonymous newborn blood spots. Punches with low enzyme activity were further evaluated by nucleotide sequence analysis of the responsible gene. Confirmation of affected infants was dependent on identification of mutations compatible with diminished enzyme activity. Results The technology for simultaneously measuring multiple enzyme activities by tandem mass spectrometry was successful. The confirmation of diagnosis for Fabry, Pompe, or MPS-I, by DNA sequencing estimated the prevalence of Fabry disease at 1/7,800 males (95% CI: 1/17,800 - 1/3,600); Pompe disease at 1/27,800 newborns (95% CI: 1/90,000 – 1/10,200); and MPS-I at 1/35,500 newborns (95% CI: 1/143,000 – 1/11,100). These estimates of prevalence are two to four times greater than the prevalence estimated by clinical diagnosis. The combined prevalence for the three disorders was 1/7,700 newborns (95% CI: 1/13,500 –1/4,500). Conclusions Tandem mass spectrometry for the simultaneous assay of multiple lysosomal enzymes can be successfully introduced into a routine newborn screening laboratory. The technology has a positive predictive value equal to, or better, than methods currently used for the detection of non-lysosomal disorders. Using newborn blood spots, the combined prevalence of Fabry, Pompe, and MPS-I is estimated at 1/7,700 newborns based on low-enzyme activity and confirmation by mutation analysis.

BackgroundThere is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life.ObjectiveTo evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases.Methods and ResultsEnzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0).DiscussionA 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively for Gram-negative Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes, as well as Gram-positive Bacillus subtilis and Staphylococcus epidermidis. Spectra have been obtained from cultures in the lag, log, and stationary growth phases excited in turn by 228.9, 244.0, and 248.2 nm light. Although Raman spectral peak positions (cm(-1)) excited by a given wavelength are very similar for all five bacterial species, the organisms are characterized by significantly different spectral intensity values. Intensity changes are associated with growth phase changes in all of the species as well. A comparison of measured with estimated average intensities has been made for spectra of log-phase E. coli. It is possible to compare measured intensities with intensities estimated for log-phase E. coli on the basis of the knowledge of its known average cellular molecular composition. A significant degree of hypochromism is observed in E. coli nucleic acid spectra. In contrast, strong average hyperchromism characterizes all aromatic amino acid peaks belonging to the same E. coli cells. Results suggest that knowledge of spectral intensity values will enhance significantly the capability to identify bacteria by means of their UV resonance Raman spectra.

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.

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Objective-To test, in a newborn screening (NBS) laboratory, the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay 5 enzymatic activities in dried blood spots (DBS) for NBS of 5 lysosomal storage diseases (mucopolysaccharidosis [MPS]-II, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII).Study design-Three mm punches from de-identified DBS were obtained from the Washington NBS laboratory and submitted to the 5-plex LC-MS/MS assay. Screen cut-offs were established by analyzing the enzymatic activity in patients confirmed to have the MPS disorder. DNA sequencing of the relevant gene was performed on a second DBS punch for all samples with enzyme activity below 10% of the mean daily activity. Results-(1)For MPS-II, 18 below cut-off samples, 1 pathogenic genotype, and 2 "high risk" genotypes; (2) For MPS-IIIB, no below cut-off samples; (3) For MPS-IVA, (4) For MPS-VI, 4 below cut-off samples and no high-risk genotypes; (5) MPS-VII, 1 below cut-off sample confirmed by genotype and clinical report to be affected.

We report a comparative study of triplex tandem mass spectrometry (MS/MS) based assays of lysosomal enzymes in dried blood spots for the early detection of Pompe, Fabry, and Hurler diseases in newborns. Four methods have been evaluated that differed in sample handling and the equipment used. A newly developed method uses assay quenching with acetonitrile to precipitate blood proteins followed by analysis on an LC-electrospray/MS/MS system capable of multiple consecutive sample injections on two parallel chromatographic columns. This method requires 1.5 min per a triplex analysis of enzyme products and internal standards, which matches the throughput of the previously reported flow injection method. LC separation reduces matrix effects and allows for more facile sample work-up. The new LC-based method showed figures of merit that were superior to those of the currently used method based on liquid-liquid extraction into ethyl acetate and flow injection into the mass spectrometer. The other methods we investigated for comprehensive comparison involved liquid-liquid extraction into ethyl acetate followed by LC-ESI/MSMS, and acetonitrile quenching followed by direct flow injection. Both methods using acetonitrile quenching were found to be robust and provide good quality data while requiring fewer liquid transfer steps and less disposable material and labor than did the extraction methods. The individual merits of the new methods are discussed to present an evaluated alternative approach to high throughput analysis in newborn screening laboratories.