Multiplex DBS enzyme assay for MPS II, IIIB, IVA, VI, VII and CLN2 via LC-MS/MS expands clinical utility of DBS enzyme testing

Enzyme analysis is the gold standard for the diagnosis of lysosomal storage disorders (LSDs). However, enzyme analysis in leukocytes requires that whole blood samples arrive to the testing laboratory within 24-48 hours of collection, and requires a relatively large volume of blood. This is problematic for the international shipment of specimens and for testing infants. The adaptation of these enzyme assays for use in dried blood spots (DBS) has ameliorated these issues for a large number of LSDs. Initially enzyme analysis in DBS exclusively utilized 4-methylumbelliferone (4-MU) fluorogenic substrates. The limitation of this methodology is that each enzyme must be measured individually. The development of tandem mass spectrometry (MS/MS) substrates has allowed multiple enzyme reactions to be combined in a single reaction, increasing efficiency. We report validation results for a new 6-plex assay for the diagnosis of five Mucopolysaccharidosis (MPS) disorders (MPS II, IIIB, IVA, VI and VII) and neuronal ceroid lipofuscinosis type 2 (CLN2) in DBS using UPLC-MS/MS. Normal reference ranges were developed using a minimum of 236 DBS samples from unaffected controls or patients with an alternate diagnosis, and 100% clinical sensitivity was established using 99 samples from patients affected with one of the six disorders. Both intra-day and inter-day precision were acceptable (