Male samples
Analysis of DBS cards was performed at a regional inherited errors of metabolism laboratory. A 3 mm diameter disc was punched from the DBS and extracted in sodium taurocholate by shaking at 4°C for 60 min. Sample extract was transferred to black 96-well microplates and incubated with substrate for 20 hours at 37°C. Samples were analysed for α-galactosidase, and acarbose-inhibited acid α-1,4-glucosidase, total acid α-1,4-glucosidase (measured as a reference enzyme) activities measured simultaneously in different wells on the same plate12. The enzymatic reactions were stopped by the addition of 100 μL/well sodium carbonate buffer (pH 9.5). Assays were performed in duplicate, and one blank was assayed for each sample. To prepare blanks, extract from each sample was added to incubated substrate solution after the enzymatic reactions were stopped. The relative fluorescence (excitation 360 nm, emission 460 nm) of each well was measured using a Biotek Synergy HT fluorescence plate reader. Fluorescence readings were corrected for the blank and compared with a 4- calibrator (supplied from Sigma)
The absolute amount of whole blood per spot is not known accurately but is comparable between samples, so enzymatic activities are expressed as pmol of substrate hydrolysed per punch per hour (pmol/punch/hour). α-galactosidase A activity below the reference range for males (6.3 – 47.0 pmol /punch/ hr) was considered significantly reduced. If activity in the first sample was low, patients were invited to return for repeat sampling and a fresh whole blood EDTA sample was sent for analysis. Repeat samples were analysed in duplicate for alpha galactosidase activity in leucocytes and plasma with beta-galactosidase and beta-hexosaminidase (A and B) analysed as reference enzymes respectively. Repeat assessment of blood spot activities of α -galactosidase and α -1,4-glucosidase was also performed. Reference ranges had been established by the local laboratory and had been validated across UK specialist laboratories. Results were collated by Queen’s Hospital, Burton and communicated to participating sites.