Quantification of glycosaminoglycans in dried blood spots, and evaluation of its usefulness as a secondary newborn screening test for mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are a group of lysosomal disorders characterized by pathological accumulation of glycosaminoglycans (GAGs). Enzyme-based newborn screening (NBS) for MPS often yields high false-positive rates because of carriers and pseudodeficient newborns. To improve screening specificity, we developed an LC–MS/MS-based enzymatic method to quantify GAG-derived disaccharides in dried blood spots (DBSs) and evaluated its utility as a second-tier test. Using DBS samples MPS-positive newborns, we quantified dermatan sulfate- (DS), heparan sulfate- (HS), and keratan sulfate-derived disaccharides. Under the primary positivity rule (DS or HS ≥ the 95th percentile cutoff), the method achieved 100% sensitivity and specificity for MPS I, completely eliminating false-positive results among MPS I carriers. For MPS II, the same rule yielded 100% sensitivity and 84.3% specificity. Reanalysis using a stricter rule requiring both DS and HS to exceed the cutoff improved specificity for MPS II to 100% without loss of sensitivity (100%). All confirmed MPS I and II patients exceeded both DS and HS cutoffs, whereas HS-only values above the cutoff occurred exclusively in MPS II pseudodeficiency, reflecting analytical and cutoff-related overlap rather than pathological GAG accumulation. Based on these findings, we recommend the stricter dual-marker rule for laboratory implementation, particularly for improving specificity in MPS II. Implementation of this approach as a second-tier test may substantially reduce false-positive referrals, eliminating all false positives for MPS I carriers and improving specificity for MPS II pseudodeficiency from 84.3% to 100%. This method may serve as a practical complementary screening tool within expanded NBS programs.