Beta-Glucosidase analysis in collected dried blood spots, report of a new method applied to the control population and patients with suspicion of Gaucher disease
Abstract
Gaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency in the enzymatic activity of β-glucosidase (BGLU), resulting in the accumulation of glucosylceramide in cells. Its diagnosis is aimed at checking the enzyme in the affected leukocytes. Studies have been conducted on dried blood spots (DBS) for BGLU activity to monitor high-risk populations; however, they exhibit interferences related to severe leukopenias or increased expression of the neutral BGLU isoform, a molecule not related to GD. This study intends to standardize a screening method on DBS (punch: 5 mm) using 4-methylumbelliferyl-β-D-glucopyranoside and chonduritol-β-epoxide. DBS samples from 395 individuals clinically suspected of GD (high-risk or HR population), 151 controls, and 16 affected patients were analyzed using the elution of 5 mm punches (≈10 μl of blood) in 300 μl of Triton X-100/ (0.5 %). As a result, the following ranges were obtained; hr: 0.84-26.92 nmol/ml/h, controls: 3.56-8.92 nmol/ml/h (M = 5.56, sd = 1.15), and patients with confirmed gd: 0.82-2.88 nmol/ml/h (M = 1.64, sd = 0.57). The cut-off point between patients with GD and controls was 3.22 nmol/ml/h, obtained from ROC analysis (99 % ci, 100 % sensitivity, and 100 % specificity). The protocol revealed a deficiency in all GD cases, confirmed by parallel BGLU analysis in isolated leukocytes. The use of CBE and the elution of 5 mm punches are recommended for enzymatic titration with a higher approximate volume of blood in the absence of neutral isoform activity.
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