Ultramicro-fluorometric assay for the diagnosis of Gaucher disease in dried blood spots on filter paper.
BACKGROUND
Gaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency of the lysosomal acid β-D-glucosidase (GBA). The aim of this study was to develop an ultramicro-fluorometric assay based on the method of Chamoles et al. for determining GBA activity in dried blood spots on filter paper (DBS).


METHODS
The assay used 3-mm diameter blood spot and 8 mmol/l of 4-methylumbelliferyl-β-D-glucoside as enzymatic substrate. The reaction occurred in plates incubated at 37°C for 20 hours and the enzyme activity was expressed in μmol hydrolysed substrate/l blood/h. The fluorescence of the enzyme product was automatically measured in a fluorometer-photometer reader (SUMA Technology).


RESULTS
The intra and inter-assay coefficients of variation were lower than 9 and 12%, respectively, and the recovery range was 97-109%.Three patients with GD were correctly diagnosed using the ultramicroassay. Healthy newborn DBS samples (n = 3003) from the National Neonatal Screening Program were analyzed, and the mean GBA activity was 5.7 μmol/l blood/h. Our assay showed high Pearson (n = 26; r = 0.99) and concordance correlations (ρc = 0.99) with the traditional method described by Chamoles et al.


CONCLUSIONS
The analytical performance characteristics of our ultramicro-fluorometric assay suggest that it can be used in the diagnosis of GD in newborns and adults.
Science and Technology Information Institute (IDICT), Miramar, Playa, Havana, Cuba.
D
D
Herrera
M
M
Monaga
D
D
Campos
Y
Y
Pampín
E C
EC
González
K
K
Lavaut
D001800:Blood Specimen Collection; D003462:Cuba / Q000453:epidemiology; D059788:Dried Blood Spot Testing / Q000379:methods; D005260:Female; D005470:Fluorometry / Q000379:methods; D005776:Gaucher Disease / Q000201:enzymology* / Q000453:epidemiology; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D015997:Neonatal Screening; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
Gaucher disease; acid-β-D-glucosidase; dried blood spots; newborn screening; ultramicro-fluorometric assay