Background: Deficiency of galactosylcerebrosidase (GALC) causes Krabbe disease. Historically, a diagnosis is made by measuring GALC enzymatic activity with a radioisotope assay. To improve the workflow and performance, we developed and clinically validated a leukocyte enzymatic assay using liquid chromatography tandem mass spectrometry (LC-MS/MS).
Materials: Extracted cell lysates were quantified and incubated with commercially available multiplexed substrates and internal standards. Liquid-liquid extraction was performed, and pre-analytical and analytical variability were evaluated and validated following clinical laboratory regulation guidelines.
Results: Enzymatic reaction products were resolved from substrate breakdown products by a 3.5-minute column separation. Intra- and inter- assay imprecision were less than 15%. No matrix effects or carryover were observed. ACD anticoagulant tubes provide the best sample stability. Detection of product was linear with an R2 of 0.99. Small differences in GALC activity were measurable near the anticipated disease range. Confirmed cases of Krabbe disease were well differentiated from carriers and non-Krabbe individuals (normal reference range).
Conclusion: An LC-MS/MS assay was developed, which can measure trace residual GALC activity in leukocytes and aid in the diagnosis of Krabbe disease. The multiplexed mixture allows for built-in sample quality control and enables a streamlined workflow for evaluation of multiple lysosomal storage diseases.
Keywords: Clinical validation; Galactocerebrosidase; Krabbe; Lysosomal storage disorder; Tandem mass spectrometry.
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