Multiplex assay for the tandem detection of ceramide trihexosides and sulfatides: Efficient first tier screening for Fabry, MLD, MSD, and Saposin B in urine

Fabry disease, metachromatic leukodystrophy (MLD), multiple sulfatase deficiency (MSD), saposin B deficiency (SAPB) and mucolipidosis II (MLII) are disorders caused by enzyme deficiencies leading to accumulation of ceramide trihexosides and/or sulfatides in tissues and increased excretion in urine. Ceramide trihexosides and sulfatides are extracted in chloroform from urine using a simple extraction method (modified from Alharbi et al, 2016) and analyzed in both positive mode for ceramide trihexosides and negative mode for sulfatides using MALDI-TOF mass spectrometry. Validation included specimens from confirmed patients affected with Fabry disease (N=16 males N=11 females), MLD (N=23), MSD (N=7), SAPB (N=2) and MLII (N=5). Fabry disease has a specific ceramide trihexoside profile pattern without presence of sulfatides. Overlapping sulfatide profiles, without ceramide trihexosides, for MLD and MSD can be confirmed by glucosaminoglycan analysis. SAPB has both the ceramide trihexoside profile pattern similar to Fabry disease and the sulfatide profile pattern similar to MLD and MSD, but reduced peak intensities. Overlapping profiles to MLII, which can have both ceramide trihexosides and sulfatides as well, can be confirmed by oligosaccharide analysis. To further delineate profile patterns, peak ratios to respective internal standards in positive and negative modes (N=30) are analyzed using a custom-made automated post-analytical multivariate pattern recognition software, Collaborative Laboratory Integrated Reports (CLIR https://clir.mayo.edu). This software generates additional ratios (N=248) between the analyte/IS ratios to aid in reliable pattern recognition.