Accurate diagnosis of lysosomal storage disorders (LSDs) such as Pompe disease and lysosomal acid lipase deficiency depends on the proper measurement of lysosomal enzyme activity through specific testing processes, according to a new study published in Genetics in Medicine.

The authors outlined new guidelines on behalf of the American College of Medical Genetics and Genomics (ACMG) to provide a resource to help implement clinical testing for these disorders based on current guidelines, review of the literature, and their expert opinions.

“This document, which focuses on clinical enzyme testing for LSDs, provides a resource for laboratories to develop and implement clinical testing, to describe variables that can influence test performance and interpretation of results, and to delineate situations for which follow-up molecular testing is warranted,” wrote the authors, who included the ACMG Laboratory Quality Assurance Committee.

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According to their standards, clinical disease features—accompanied by abnormal results from parental carrier screening, prenatal screening following chorionic villus sampling or amniocentesis, newborn screening, urine screening, and/or molecular testing—warrant lysosomal enzyme testing to confirm an LSD.

Read more about Pompe disease diagnosis

Preanalytical requirements included sample types for lysosomal enzyme activity assays: dried blood spots, leukocytes, serum/plasma, and fibroblasts. Newborn screening uses dried blood spots to detect conditions like Pompe disease which are listed on the United States Health and Human Services’ Recommended Uniform Screening Panel.

Heat, extreme cold, repeated freezing and thawing processes, humidity, and dampness all interfere with enzyme activity levels, influencing the final assay results, the authors said. Sample processing using centrifugation and other methods must occur as soon as possible after collection to prevent denaturing of the enzymes and to stabilize enzyme activity levels.

When clinicians suspect an LSD, 3 samples must be processed for comparison—a positive control from an individual already diagnosed with the suspected LSD, a normal control from an unaffected individual, and a sample from the individual being screened. The guidelines specify assay conditions (which differ for each LSD) including buffer and substrate concentrations, pH levels, protein concentration ranges, and duration and temperature for incubation.

For enzyme analysis, they recommend samples with abnormal enzyme activity levels undergo further genetic sequencing and specific biomarker testing to differentiate a true LSD from pseudodeficiencies exhibiting low enzyme activity levels unrelated to clinical disease.

Lysosomal enzyme activity should be assayed using digital microfluidics fluorimetry or tandem mass spectrometry, such as flow injection analysis or liquid chromatography. Laboratory technicians must report any interference or imprecisions during testing. Lastly, the guidelines establish current reference ranges for known LSDs, indicating unaffected individuals, affected individuals, heterozygous individuals, or inconclusive results.


Strovel ET, Cusmano-Ozog K, Wood T, et al. Measurement of lysosomal enzyme activities: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med. Published online February 10, 2022. doi:10.1016/j.gim.2021.12.013