Test ID: BGABS    
Beta-Galactosidase, Blood Spot

Whole blood collected in ACD or EDTA anticoagulant tubes is spotted onto filter paper. A 1/8-inch (3-mm) disk is punched out of the dried blood spot (DBS) into a 96-well, round-bottom plate containing 40 microliters of 50 mM cit-phos buffer as elution liquid and 20 microliters of 0.8 mM 4-methylumbelliferyl-beta-D-galactopyranoside in water as the substrate (60 microliters total volume + DBS). A blank is prepared using only elution liquid, substrate, and filter paper punches containing no blood (60 microliters total volume + blank punches). All patients, controls, and blank are set up in duplicate (2 punches total, 1 punch per well). After the incubation period (3 hours at 37 degrees C), all of the liquid from the plate is manually transferred to a 96-well, flat-bottom black plate. A calibration curve is prepared and analyzed on every plate to calculate enzyme activity results, based on fluorescence units in patient wells vs. calibrators. The calibration is derived from 4-methylumbelliferone (4-MU) that is serially diluted manually in the plate with the highest calibrator being equivalent to an enzyme activity of 10.4 nmol/hour/mL. Two hundred microliters of stop buffer (150 mM EDTA, pH 11.4) is added to all wells (patients, QC, blanks, calibrators). The plate is then read on the spectrofluorometer. Fluorescence readings for duplicate wells are averaged, and the average fluorescence is used to calculate the enzyme activity result.(Civallero G, Michelin K, de Mari J, et al: Twelve different enzyme assays on dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases. Clin Chim Acta 2006;372:98-102)

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