Background: Fabry disease is a progressive, X chromosome-linked lysosomal storage
disorder with multiple organ dysfunction. Due to the absence or reduced activity of
alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide
(Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous
and currently routinely used fluorometry-based assays measuring mean activity mostly
fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry
assay to measure AGAL activity in individual cells. Methods: Conventional and multispectral
imaging flow cytometry was used to detect AGAL activity. Specificity was validated
using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin.
The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with
exome sequencing, gene expression and substrate accumulation. Results: Flow cytometric
detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In
the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation
are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan
blue solution is necessary for the specific detection of lysosomal substrate accumulation.
Conclusion: A flow cytometry-based assay was developed for the quantitative detection
of AGAL activity at the single-cell level, which may contribute to the diagnosis of
Fabry patients.
