ABSTRACT
BACKGROUND: The goal of this study was to assess the biochemical parameters of the enzymes α-l-iduronidase (IDUA) and arylsulfatase B (ASB), which are deficient in mucopolysaccharidosis (MPS) I and VI, respectively, in dried blood spot (DBS) samples impregnated on filter paper. METHODS AND RESULTS: The optimal pH, Km, and Vmax of IDUA and ASB in DBS are hereby presented. After these analyses, the reference values for the activities of these enzymes in DBS with cutoff of 3.65nmol/h/mL for IDUA and 6.80nmol/h/mL for ASB were established. The research also showed that the stability (21days) of the IDUA activity is lower than ASB, which maintained its enzymatic activity stable up until 60days of analysis, after impregnating the filter paper with blood. CONCLUSION: Currently, DBS ensures important advantages in handling storage and transportation of samples with respect to neonatal screening programs. This study contributes to characterizing and differentiating the biochemistry of deficient enzymes in MPSs I and VI of DBS samples.
Subject(s)
Dried Blood Spot Testing/methods , Iduronidase/blood , Mucopolysaccharidosis IV/blood , Mucopolysaccharidosis I/blood , N-Acetylgalactosamine-4-Sulfatase/blood , Dried Blood Spot Testing/instrumentation , Female , Humans , MaleABSTRACT
BACKGROUND: Due to the importance and the difficulty still present in determining the biochemical diagnosis of Fabry disease (FD), the aim of this study was to establish and compare the biochemical and kinetic properties of alpha-galactosidase A (GLA) in dried blood spots (DBS), plasma and leukocyte samples of FD patients and healthy subjects to evaluate the possible use of these parameters as an auxiliary tool in the diagnosis of this disease. METHODS: GLA activity in DBS, plasma and leukocyte samples from Fabry disease patients and healthy subjects was compared and characterized in terms of optimal pH, Km and Vmax and heat stability. RESULTS: A difference was observed between the Km and Vmax of FD patients and healthy controls using DBS, plasma and leukocyte samples. In leukocytes, pre-incubation at 50°C for 60 min was effective to differentiate FD patients from healthy controls. CONCLUSION: These results can be used as an auxiliary method to the FD diagnosis, especially in cases of patients whose GLA activity is within normal range.
Subject(s)
Fabry Disease/blood , Fabry Disease/diagnosis , Leukocytes, Mononuclear/enzymology , alpha-Galactosidase/metabolism , Case-Control Studies , Dried Blood Spot Testing , Enzyme Stability , Fabry Disease/pathology , Female , Hot Temperature , Humans , Kinetics , Leukocytes, Mononuclear/pathology , MaleABSTRACT
This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.
Subject(s)
Hydrolases/metabolism , Lysosomal Storage Diseases/diagnosis , Lysosomes/enzymology , Lysosomes/metabolism , Blood Specimen Collection , Female , Humans , Lysosomal Storage Diseases/enzymology , Lysosomal Storage Diseases/metabolism , Male , Mass Screening/methodsABSTRACT
This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and ß-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis.
Subject(s)
Biological Assay , Gaucher Disease/blood , Gaucher Disease/diagnosis , Hexosaminidases/blood , Leukocytes/metabolism , Mass Screening , beta-Galactosidase/blood , Blood Specimen Collection , Brazil , Case-Control Studies , HumansABSTRACT
OBJECTIVES: To compare alpha-galactosidase A activity in dried blood spots on filter paper, plasma, and leukocytes of Fabry disease patients and healthy controls, and to develop a miniaturization approach of the techniques to measure activity using plasma and leukocytes. DESIGN AND METHODS: Blood was collected from healthy controls and Fabry disease patients. Two drops were spotted on filter paper. Plasma and leukocytes were separated from the remaining sample. Enzyme activity was assessed by fluorometry. RESULTS: Significant positive correlation between standard and miniaturized techniques was observed. Alpha-galactosidase activity differed for male and female subjects when analyzed using filter paper and plasma. New reference and cutoff values were established based on the differences in alpha-galactosidase activity between genders. A good correlation was observed across biological materials assessed. CONCLUSIONS: The establishment of specific values for men and women increases reliability of commonly used techniques to screen and diagnose Fabry disease.
Subject(s)
Fabry Disease/blood , Leukocytes/enzymology , alpha-Galactosidase/blood , Adolescent , Adult , Blood Specimen Collection , Case-Control Studies , Dried Blood Spot Testing , Fabry Disease/diagnosis , Fabry Disease/enzymology , Female , Humans , Male , Paper , Plasma , Reference Values , Sensitivity and Specificity , Young AdultABSTRACT
OBJECTIVES: To analyze the effect of blood collection and storage conditions on activity of α-galactosidase A, arylsulfatase B and α-glucosidase. DESIGN AND METHODS: Blood was collected in EDTA, heparin, or direct spotting on filter paper and stored at different temperatures (-20, 4, 25 and 37°C) and storage times (3, 10, 17 and 180 days). The influence of filter paper size was also assessed (3.0 and 1.2mm). RESULTS: No statistically significant difference was observed between the three collection methods. α-Glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used. CONCLUSIONS: Both paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection.
Subject(s)
Blood Specimen Collection/methods , Filtration , N-Acetylgalactosamine-4-Sulfatase/blood , Paper , alpha-Galactosidase/blood , alpha-Glucosidases/blood , Humans , Temperature , Time FactorsABSTRACT
BACKGROUND: Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. METHODS: In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for ß-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. RESULTS: Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of ß-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. CONCLUSIONS: The two enzymes investigated in the present study may be stored for up to 17 days (ß-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.