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Background Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha galactosidase A (AGAL, EC 3.2.1.22). Despite increasing utilization of dried blood spot (DBS) as samples for AGAL enzyme assays, the effects of blood sample collection techniques on enzyme activity have not been studied. Methods DBS samples were prepared by spotting blood collected into an ethylenediaminetetraacetic acid (EDTA) tube and by direct application of blood from a finger prick or a venipuncture syringe. AGAL activity was measured quantitatively by detecting the fluorescence of 4-methylumbelliferone (4-MU) generated using the substrate 4-methylumbelliferyl-α- d -glucopyranoside (4-MUGal) in an acidic pH for 20 h. N-acetyl- d -galactosamine (GalNAc) was used to inhibit α-galactosidase B (EC 3.2.1.49). Results We studied 88 previously diagnosed Fabry disease patients and 690 healthy controls. Average AGAL activity in DBS samples prepared using EDTA tubes was higher compared to those spotted directly irrespective of disease status. Conclusions The study confirms the need for collection method-specific reference ranges using DBS samples.