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.