An enzyme immunoassay for determining 17alpha-hydroxyprogesterone in dried blood spots on filter paper using an ultramicroanalytical system.

BACKGROUND: 17alpha-hydroxyprogesterone has been used for the diagnosis of congenital adrenal hyperplasia (CAH) in the newborn period. METHODS: A simple and rapid competitive ultramicro ELISA assay based on competition between 17-OHP-alkaline phosphatase conjugate and 17-OHP in blood specimens for a limited number of binding sites on specific polyclonal rabbit anti-17-OHP antibodies, has been developed for the measurement of 17-OHP in dried blood spots on filter paper. The assay buffer contains danazol to displace 17-OHP from steroid-binding proteins. RESULTS: The 17-OHP assay was completed in 3 h, with a measuring range of 10-250 nmol/l. The intra- and inter-assay CV were 5.5-8.2% and 6.4-9.1%, respectively, depending on the 17-OHP concentrations. The recovery ranged from 98-103%. Of 3750 newborn samples collected on filter paper, 903 from the national neonatal screening program were analyzed, and the mean 17-OHP concentration was 12.2 nmol/l. Our assay showed high Pearson and concordance correlations with the commercially available ICN Neoscreen ELISA 17alpha-hydroxyprogesterone kit. CONCLUSIONS: The analytical performance characteristics of our 17-OHP Neonatal UMELISA suggest that it can be used for the neonatal screening of CAH.

Qualitative colorimetric ultramicroassay for the detection of biotinidase deficiency in newborns.

BACKGROUND: We describe a simple qualitative visual ultramicroassay based on the colorimetric method introduced by Heard et al. for the detection of biotinidase deficiency in dried blood samples spotted on filter paper. METHODS: The assay uses 3-mm discs of dried blood on Schleicher and Schuell 903 filter paper and ultramicrovolumes of each reagent. Ten thousand newborn samples from the National Screening Program for the Detection of Phenylketonuria were evaluated. RESULTS: The ultramicroassay shows a good reproducibility. The lower detection limit is around 2% of the mean normal activity. We found one sample with the absence of enzymatic activity, another that was between 10% and 30%, and 10 with activity levels <40%. There was coincidence of our results with those obtained by the conventional colorimetric method that uses B-PAB as substrate. CONCLUSIONS: The qualitative colorimetric ultramicroassay does not require special laboratory equipment and it is suitable for the neonatal screening of biotinidase deficiency.

Medición de TSH, T4 y Phe en muestras de sangre del cordón umbilical en papel de filtro: impacto en el tamizaje neonatal / Measurements of TSH, T4 and Phe in samples of umbilical cord blood on filter paper: impact on neonatal screening

En este momento existe consenso en que el hipotiroidismo congénito y la fenilcetonuria constituyen modelos teóricos prácticos ideales en el tamizaje neonatal. La tendencia actual en el mundo es la salida temprana de las maternidades debido a razones socioeconómicas, lo cual guía, sin duda, a la implementación de estos programas en sangre del cordón umbilical. Se evaluaron los niveles de T4 total, TSH y Phe en muestras de sangre del cordón umbilical de 458 recién nacidos, colectadas en papel de filtro S&S 2992, utilizando ultramicroensayos fluorescentes. La distribución de frecuencias para las concentraciones de TSH mostró un valor medio de 1,7 mUl/l; el 98 por ciento de las muestras tuvieron concentraciones en el rango de 0-10 mUl/l, el 1,7 por ciento entre 10-20 mUl/l y una muestra con valor mayor de 20 mUl/l; para T4 el valor medio de concentración fue de 177,2 nmol/l; el 90,2 por ciento tuvo valores entre 116-370 nmol/l, el 9,6 por ciento entre 60-116 nmol/l y una muestra con un valor menor de 60 nmol/l. Para Phe, el valor promedio fue de 4,6 µmol/l (0,7mg/dl), el 80,3 por ciento entre 0-60 µmol/l (0-1 mg/dl), el 19,2 por ciento entre 60-120 µmol/l (1-2 mg/dl) y 2 muestras con valor mayor de 120 µmol/l ( 2 mg/dl). Los resultados obtenidos demuestran que es factible el empleo de muestras en sangre del cordón para el diagnóstico precoz de hipotiroidismo congénito, no así en el caso de la Phe donde los niveles detectados fueron muy bajos por lo que no garantiza total efectividad en el diagnóstico de fenilcetonuria