We have developed a tandem mass spectrometry based assay of iduronate-2-sulfatase (IdS) activity for the neonatal detection of mucopolysaccharidosis II (MPS-II, Hunter Syndrome). The assay uses a newly designed synthetic substrate (IdS-S) consisting of α-L-iduronate-2-sulfate, which is glycosidically conjugated to a coumarin and a linker containing a tert-butyloxycarbamido group. A short synthesis of the substrate has been developed that has the potential of being scaled to multigram quantities. Sulfate hydrolysis of IdS-S by IdS found within a 3 mm dried blood spot specifically produces a nonsulfated product (IdS-P) which is detected by electrospray tandem mass spectrometry and quantified using a deuterium-labeled internal standard, both carried out in positive ion mode. Analysis of DBS from 75 random human newborns showed IdS activities in the range of 4.8–16.2 (mean 9.1) μmol/(h L of blood), which were clearly distinguished from the activities measured for 14 MPS-II patients at 0.17–0.52 (mean 0.29) μmol/(h L of blood). The assay shows low blank activity, 0.15 ± 0.03 μmol/(h L of blood). The within-assay coefficient of variation (CV) was 3.1% while the interassay CV was 15%.

We report a new assay of N-acetylgalactosamine-4-sulfatase (aryl sulfatase B) activity in dried blood spots (DBS) for the early detection of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) in newborn screening. The assay uses a synthetic substrate consisting of N-acetylgalactosamine-4-sulfate moiety glycosidically linked to a hydrophobic residue and furnished with a tert-butyloxycarbamido group as a marker for specific mass spectrometric fragmentation. Incubation with aryl sulfatase B present in DBS converts the substrate to a desulfated product which is detected by electrospray tandem mass spectrometry and quantified using a homologous internal standard. Assay and work-up procedures were optimized to be compatible with the work flow in newborn screening laboratories. Analysis of DBS from human newborns showed clear distinction of aryl sulfatase B activity from 89 healthy individuals where it ranged between 1.4–16.9 μmol/(h × L blood), with an average activity of 7.4 μmol/hr/L blood, and an MPS-VI patient that had an activity of 0.12 μmol/(h × L blood). Results are also reported for the aryl sulfatase B assay in DBS from groups of normal felines and felines affected with MPS-VI.

The clinical phenotype of Sanfilippo Syndrome is caused by one of four enzyme deficiencies that are associated with a defect in mucopolysaccharide metabolism. The four subtypes (A, B, C, and D) are each caused by an enzyme deficiency involved in the degradation of heparan sulfate. We have developed a highly efficient synthesis of the substrates and internal standards required for the enzymatic assay of each of the four enzymes. The synthesis of the substrates involves chemical modification of a common intermediate. The substrates and internal standards allow the measurement of the enzymes relevant to heparan N-sulfatase (type A); N-acetyl-α-glucosaminidase (type B); acetyl-CoA:α-glucosamide N-acetyltransferase (type C); N-acetylglucosamine 6-sulfatase (type D). The internal standards are similar to the substrates and allow for the accurate quantitation of the enzyme assays using tandem mass spectrometry. The synthetic substrates incorporate a coumarin moiety and can also be used in fluorometric enzyme assays. We confirm that all four substrates can detect the appropriate Sanfilippo syndrome in fibroblast lysates, and the measured enzyme activities are distinctly lower by a factor of 10 when compared to fibroblast lysates from unaffected persons.

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BackgroundManufacturing methods for dimethyl sulfoxide (DMSO)‐cryopreserved platelets (CPPs) are manual and labor intensive. Thawing and prepare‐for‐transfusion steps are in an open system that requires transfusion within 4 h. A fill‐and‐finish system (CUE) can automate the manufacturing process. A newly configured bag system allows freezing, thawing, and use of resuspension solutions while maintaining the functionally closed system, and extending the post‐thaw shelf life beyond 4 h. Our objective is to evaluate the feasibility of the CUE system and the functionally closed bag system.Study design and MethodsDMSO was volumetrically added to double‐dose apheresis platelets, concentrated, and delivered to a 50‐ or 500‐mL ethylene‐vinyl acetate (EVA) bag by the CUE (n = 12). The functionally closed bag system contained 25 mL platelet additive solution 3 (PAS‐3) in a 50‐mL EVA bag. Control CPP (n = 2) were manually prepared. PAS‐3 and CPP were thawed together. CPP were stored up to 98 h (20–24°C) and tested using a standard assay panel.ResultsCUE prepared CPP met the design targets: volume, platelet content, and DMSO concentration. CUE CPP P‐selectin was high. CD42b, phosphatidylserine (PS) expression, and live cell percentage were favorable compared to controls and favorably maintained over storage. The thrombin generation potency was slightly reduced compared to controls. The 50 mL EVA bag maintained pH for up to 30 h, and the 500 mL EVA bag beyond 76 h.DiscussionThe CUE system presents a technically feasible method to prepare CPP. A functionally closed bag system with resuspension solution was successful and can extend the post‐thaw storage time of CPP.