B-204 Determination of Lyso-GB3 in Dried Blood Spots: A Useful Biomarker for Fabry Disease

Abstract

Abstract Background Fabry disease, an X-linked inborn error of metabolism, results from pathogenic mutations in the α-galactosidase A gene (GLA). These mutations reduce or abolish the α-galactosidase A activity, which result in accumulation of glycosphingolipids in the lysosomes, including globotriaosyl-sphingosinein (lyso-GB3), throughout the body, in particular the kidney, heart, and brain. Lyso-GB3 may be elevated in symptomatic patients and supports a diagnosis of Fabry disease. It may also be helpful as a tool for monitoring disease progression, as well as determining treatment response in known patients. Moreover, measurement of Lyso-GB3, may provide additional diagnostic information in the evaluation of uncertain cases, such as in asymptomatic heterozygous female patients, individuals with novel GLA variants of unclear clinical significance, as well as asymptomatic patients identified by family screening. This study aimed to determine Lyso-GB3 in dried blood spot (DBS) samples with a simple, rapid and sensitive LC-MS/MS method, and identify its reference interval in the Brazilian population. Methods All experiments were performed on the Waters TQ-S Micro LC-MS/MS system. The gradient chromatographic separation was carried out using a Waters BEH C18 column in 3.5 min. One DBS punch of 3.1 mm diameter was used for analysis in a microtiter plate and then, a solvent extraction containing stable isotope analogue of Lyso-GB3 was added. After incubation time, the supernatant was directly injected into LC-MS/MS system. The method was validated for linearity, carryover, intra and inter-day precision and the limit of quantification was determined. A reference interval was obtained by analyzing 94 healthy volunteers (49 women and 45 men). The difference of Lyso-GB3 concentration between men and women was investigated (Wilcoxon paired test). The reference interval was determined by the superior limit of the confidence interval (90% CI) of a normal distribution of the results. Results The method was linear between 0.2–100.0 ng.mL−1. The recovery was between 81.0 and 139.7%, which was acceptable considering CLSI guidelines for DBS validation (CLSI NBS04-A). The intra and inter-day precision were below 12.1%, and the limit of quantification was 0.2 ng.mL−1. The reference interval determined was below 0.8 ng/mL. Conclusion In conclusion, the LC-MS/MS method for Lyso-GB3 determination in DBS samples was successfully validated. The sample preparation is simple and the chromatographic method is rapid for routine laboratory. The cutoff determined is comparable with what is available in the literature and adequate for analysis in clinical routine.