Whole Blood alpha-D-Galactosidase A activity for the identification of Fabry patients

Rationale: Fabry disease is a rare X-linked lysosomal storage disorder due to deficiency of α-D-galactosidase A (α-Gal A) and the consequent progressive accumulation of globotriaosyl ceramide in plasma and lysosomes of all tissues. Particularly, accumulation in the kidney and heart results in early mortality of hemizygous males; the syndrome affects also the heterozygous females. The availability of enzymatic replacement therapy for Fabry disease urges the need of a reliable, sensitive and routinely standardised assay to reach a prompt diagnosis for selected patients with neurological , cardiological and kidney damage. Methods: Alpha-D-galactosidase A activity was determined in heparinised whole blood form 30 healthy individuals, 7 hemyzigous males and 7 heterozygous females affected by Fabry disease. We used 50 L-samples and we determined the enzymatic activity by means of a fluorimetric method. To show the validity of the whole heparinised blood assay, we compared the results with those obtained from leukocytes, plasma and dried blood spot collected from the same individuals. Results: The analytical characteristics of the whole blood method are: linearity up to 2000 mU/L with R=0.998; detection limit of 4 mU/L; enzymatic stability for 9 days at 4°C; total analytical imprecision ranging from 3.27 to 5.72 coefficient of variation; less than 10% for haemoglobin and bilirubin interference effect. The correct identification of Fabry patients is: all hemyzigous males and 5 heterozygous females were correctly identified. Conclusion: Fluorimetric determination on whole blood sample of alpha-D-galactosidase A activity was shown to be a sensitive and reliable method when compared with activity determination from leukocytes, plasma and dried blood spot. We propose our method as a routinely standardised assay to reach a prompt diagnosis of Fabry disease in selected risk populations (patients presenting with stroke, hypertrophic cardiomyopathy or dialytic patients).