Neonatal screening for mucopolysaccharidoses by determination of glycosaminoglycans in the eluate of urine‐impregnated paper: preliminary results of an improved DMB‐based procedure

Abstract

The fact that mucopolysaccharidoses (MPSes) are now treatable, and that the earlier treatment is initiated the better, is an indication for neonatal screening. The most efficient approach seems likely to be a multi-tier procedure in which screening for urinary glycosaminoglycan (GAG) is followed by enzyme determinations in heelprick blood of newborns screening positive. Hitherto the method of choice for the determination of GAG has been the measurement of absorbance by a complex of GAG and 1,9-dimethylmethylene blue (DMB). We evaluated a DMB method in which absorbance by DMB is measured following its addition to the eluate obtained from paper-borne newborn urine samples and is normalized relative to urinary creatinine. Calibration is performed with chondroitin-6-sulfate (Ch-6-S). The limits of detection and quantification of GAG were 1.98 and 5.94 mg/dl, respectively. The within-run coefficients of variation (CVs) of the GAG/creatinine ratio for 25, 31, and 70 mg/dl solutions of Ch-6-S in urine were 21.8, 16.4, and 10.5%, respectively, and the corresponding between-run CVs were 25.0, 13.5, and 10.1%. Recovery from the urine spiked with 31 mg Ch-6-S/dl was 94.8%. Accuracy was also acceptable for all other GAGs except hyaluronic acid. For neonatal screening, the diagnostic threshold was tentatively established as 800 mg GAG/g creatinine, the 95th centile of samples from 903 infants aged 3-28 days, but the value of the GAG/creatinine ratio was negatively correlated with age. Application of the new method to samples from older individuals with and without MPS achieved 100% sensitivity and specificity when used with an age-dependent threshold taken from the literature on the original DMB method. If used in the first tier of a multi-tier screening protocol, the proposed method would allow the detection of abnormal levels of all GAGs except hyaluronic acid.