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Newly Developed Ultra-High-Accuracy Assays Help in Post-Screening Diagnosis of Lysosomal Diseases

Dr. Gelb - Speaker World Symposium 2017Michael H. Gelb, MD

February 18, 2017—San Diego, California—Ultra-high-accuracy combination liquid chromatography + mass spectrometry assays of galactosylceramidase, acid alpha-glucosidase, and alpha-L- iduronidase have been developed that help in follow-up diagnosis of Krabbe, Pompe, and mucopolysaccharidosis type I, respectively.

This description of newly developed, ultra-high-accuracy assays was presented at the 13th Annual WORLDSymposium, from February 13–18.

Michael H. Gelb, MD, of the University of Washington, Seattle, explained that newborn screening for lysosomal diseases requires second-tier analysis to determine the likely severity. Tandem mass spectrometry for multiplex assay of enzymatic activities in dried blood spots has emerged as the major method of newborn screening of lysosomal diseases.

Pilot studies in Washington state, Kentucky, and New York have shown that the rate of false–positives is estimated to be three- to fivefold lower than the rate provided by fluorimetric methods including digital microfluidics when equivalent cut-off values are used.

Using lymphocytes, the tandem mass spectrometry multiplex assays are carried out for galactosylceramidase, acid alpha-glucosidase, and alpha-L-iduronidase for follow-up diagnosis of Krabbe, Pompe, and mucopolysaccharidosis I diseases, respectively.

Mass spectrometry has also been developed to screen for additional lysosomal diseases (mucopolysaccharidosis types II, IIIB, IVA, VI, and VII diseases, ceroid lipofuscinosis types I and II, metachromatic leukodystrophy, and lysosomal acid lipase deficiency).

The assay is being piloted in the Washington state newborn screening lab, and data on approximately 40,000 babies has been collected. The assay is very robust to date, with a manageable number of false-positives.

A third pilot study has been initiated for metachromatic leukodystrophy, cerebrotendinous xanthomatosis, and type C Niemann-Pick disease. This third pilot study also involves quantification of biomarkers in dried blood spots using tandem mass spectrometry. Data on the first 8000 babies look promising.

The data show that the severity of lysosomal diseases correlates well with the level of residual enzymatic activity. Differences between affected and non-affected (pseudodeficiency) patients are due to small differences in residual enzymatic activity, but these can be readily distinguished by using liquid chromatography tandem mass spectrometry analysis in leukocytes prepared from a small volume of venous blood (<0.5 mL).

Dr. Gelb concluded that the two key advantages of the tandem mass spectrometry assays his team has developed is that it provides a large analytical range, that is, a high span of enzymatic activity values vs other methods such as fluorimetric and radiometric enzyme assays.

The second key feature is that several lysosomal storage diseases cannot be evaluated by fluorimetric methods, yet all can be evaluated using mass spectrometry.

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Molecular Genetics and Metabolism is a contribution to the understanding of the metabolic basis of disease. The journal publishes articles describing investigations that use the tools of biochemistry and molecular biology for studies of normal and diseased states. In addition to original research articles, occasional minireviews reporting timely advances as well as brief communications and letters to the editor are considered.


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