Cuban neonatal screening of phenylketonuria using an ultramicro-fluorometric test

Immunoassay Center, 134th Street and 25th Avenue, Postal Code 6653, Cubanacan, Playa, Havana, Cuba.
Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.82). 04/2009; 402(1-2):129-32. DOI: 10.1016/j.cca.2008.12.039
Source: PubMed


Guthrie's bacterial inhibition assay was used in Cuba, since 1983. A decentralized program for the newborn screening of hyperphenylalaninemias started in the year 2000 using an ultramicro-fluorometric test (UMTEST PKU).
A simple and rapid ultramicro-fluorometric test based on McCaman and Robin's method has been designed, developed and applied for the measurement of Phe in dried blood spots on filter paper.
The UMTEST PKU exhibited an acceptable precision and accuracy. Samples of 27528 newborns on filter paper Schleicher & Schuell 903 (S&S 903) from the National neonatal screening program were collected and analyzed, and the mean Phe concentration was 66.5 micromol/l. Our assay showed high Pearson and concordance correlations with 2 commercially available kits. A total of 521923 Cuban newborns were studied from the year 2000 to 2007 using the UMTEST PKU. Elevated blood phenylalanine levels were found in 1764 infants (0.34%) and no false negative were noted. Ten cases were diagnosed with phenylketonuria, all of them with an initial phenylalanine concentration over 360 micromol/l.
The analytical performance characteristics of our assay and its use in the National program have demonstrated its suitability for the neonatal screening of PKU.

31 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: To describe a simple, rapid, quantitative ultramicrotest (UMTEST) based on the fluorometric method introduced by Fujimura et al. adapted to an Ultra Micro Analytic System (SUMA) for the detection of total galactose (GAL) in dried blood specimens. The assay uses 3 mm discs of dried blood on Whatman 903 filter paper and small volumes of each reagent. A methanol/acetone/water solution is used for deproteination, and a specially designed 96-well polystyrene opaque ultramicroplates, with a maximum capacity of 30 μL per well, are used for the reading. The UMTEST GAL is completed in 2 h, with measuring range of 0.28-3.92 mmol/L. The intra- and inter-assay coefficients of variation were 2.3%-8.9% and 6.8%-11.1%, respectively, depending on the total GAL concentrations. Percentage recovery ranged from 97.7% to 103%. Limit of detection and limit of quantitation were 0.06 and 0.16 mmol/L, respectively. The mean GAL concentration, in 2510 dried blood samples from the National Neonatal Screening Program was 0.23 mmol/L. Our assay showed high concordance correlations with the commercially available ICN Immuno-Chem™ GAL-MW EA kit. The analytical performance characteristics of this assay is suitable for mass newborn screening of galactosemia in Cuba.
    No preview · Article · Jan 2011 · Journal of Perinatal Medicine
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The aims of the study were to prepare macro peptides low in phenylalanine (Phe) from non-conventional raw materials, and to demonstrate the feasibility of using the fluorometric technique to measure the diminution of their Phe content. Aqueous solution of flours of legumes, and amaranth panicles were used to elaborate the concentrates by using isoelectric precipitation. These protein concentrates, and a whey solution were incubated with proteolytic enzymes to hydrolyze the peptide link at the aromatic amino acids, and then these macro peptides were filtrated through activated charcoal, in order to reduce its phenylalanine concentration. The Phe concentration, of the each prepared macro peptides, was analyzed by using fluorometric technique, and it was later validated by using HPLC. The crude protein contents in the concentrates have varied from 90% in the protein isolate from lentils, 76% in those from the frijol white, and 44% in those from amaranth panicles. Protein concentrates, and whey were hydrolyzed by using the following enzymes: pepsin from the pig gastric mucosa, protease from Aspergillus oryzae, and protease type XIV from Streptomyces griseus. It was determined that the enzymes with the better hydrolysis capacity, were the proteases from S. griseus and A. oryzae. The macro peptides with non-linked phe were filtered through activated charcoal. Reductions of Phe of up to 99% in the second and third filtrate were observed and this reduction was corroborated by using HPLC technique. It was also established the higher sensitivity of the fluorometric method to detect Phe, than the HPLC technique.
    Full-text · Article · Jun 2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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). 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). 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. The analytical performance characteristics of our ultramicro-fluorometric assay suggest that it can be used in the diagnosis of GD in newborns and adults.
    No preview · Article · Nov 2013
Show more