High-Resolution Mass Spectrometry Method for Targeted Screening and Monitoring of Fabry, Gaucher and ASMD Using Dried Blood Spots and Capitainers: Impact of Sample Matrix on Measurement Results

Abstract

The sphingolipidoses Fabry disease, Gaucher disease and Acid sphingomyelinase deficiency (ASMD) are the three most common lysosomal storage diseases for which treatment is currently available. Timely diagnosis with estimation of the disease severity and possibilities of follow-up of patients, whether or not under therapy, is crucial for providing good care and for the prevention of possible lethal complications. With this research we provide an efficient and sensitive detection method; its implementation in clinical practice could optimize the diagnosis and follow-up of patients with Gaucher, Fabry and ASMD. This detection method on dried blood spots (DBS) was validated according to the international Clinical and Laboratory Standards Institute (CLSI) guidelines, looking at reproducibility, linearity, carry-over and lower limit of quantification. Analogously, validation and subsequent comparison of the method validation results using another matrix, the Capitainer blood sampling cards (Capitainers), was fulfilled. The results showed that this detection method is fully applicable clinically when using DBS as well as Capitainers. In addition, even additional improvements of some validation parameters were found when using the Capitainers. Twenty-six patient samples and fifteen healthy samples were analyzed for case finding control. All patient cases were detected without ambiguity. We present a high-resolution mass spectrometry method that provides an accurate analysis for targeted screening, aiming for improved/accelerated diagnosis when added in the diagnostic pathway and monitoring of Fabry, Gaucher and ASMD in DBS as well as in Capitainers, with the main advantages of a small volume of blood samples, guaranteeing stability and easy transportation from the collection site to the laboratory.

Keywords: ASMD; Fabry; Gaucher; GlcSph; LC-MS/MS; Lyso-Gb1; Lyso-Gb3; Lyso-SM; Lysosomal storage disease; biomarker; capitainer; dried blot spot; filter system; screening; sphingolipidoses.

Figure 1

Diagram depicting the clinical symptomatology of Gaucher disease, Fabry disease and ASMD: shared and distinct features in lysosomal storage disorders. This diagram contains the typical clinical symptoms of Gaucher disease, Fabry disease and ASMD [9,10,15,16,17,19,20,21,22]. Disease-specific symptoms that are unique to one of the three disorders are noted inside the corresponding circle. Overlapping clinical symptoms between two disorders are listed in the boxes connected with an arrow to the relevant overlapping sections. The starred box lists the symptoms common to all three disorders. The gray boxes indicate the specific enzyme deficiency and associated lysosphingolipid biomarker for each specific disorder.

Figure 2

(A–C): Linearity of the calibration curve in DBS. Based on 7 standard values, linearity is shown for each biomarker by comparing the spiked concentrations with the effectively measured concentrations. Concentration units are expressed in ng/mL (or 10⁻⁶ g/L). The * stands for the x in the regression equation, see Table 1. (A) Bland–Altman curve for GlcSph. (B) Bland–Altman curve for Lyso-Gb3. (C) Bland–Altman curve for Lyso-SM.

Figure 3

(A–C): Linearity of the calibration curve in CAP. Based on 7 standard values, linearity is shown for each biomarker by comparing the spiked concentrations with the effectively measured concentrations. Concentration units are expressed in ng/mL (or 10⁻⁶ g/L). The * stands for the x in the regression equation, see Table 1. (A) Bland–Altman curve for GlcSph. (B) Bland–Altman curve for Lyso-Gb3. (C) Bland–Altman curve for Lyso-SM.

Figure 4

Boxplot representing the biomarker levels in each sample group. The red dots are the individual measurement results, which can be found in Table 6.