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Clin Chem. 2017 Apr;63(4):842-851. doi: 10.1373/clinchem.2016.259036. Epub 2017 Feb 14.

Liquid Chromatography-Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease.

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Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY.
Laboratory of Human Genetics, New York State Department of Health, Wadsworth Center, Albany, NY.
Departments of Chemistry and.
Biochemistry, University of Washington, Seattle, WA.
Newborn Screening Translation Research Initiative, National Foundation for the Centers for Disease Control and Prevention, Inc., Atlanta, GA.
Departments of Chemistry and
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY;



Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual's phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples.


We incubated 10 μL leukocyte lysate and 25 μL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid-liquid extraction. The extracts were evaporated and reconstituted in 200 μL methanol and analyzed by LC-MS/MS for GAA activity.


A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%-1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44-1.75 nmol · h-1 · mg-1 and 2.0-6.5 nmol · h-1 · mg-1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0-28.1 nmol · h-1 · mg-1, showing substantial but incomplete separation from the LOPD group.


This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.

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