RESUMEN
PURPOSE: New therapies for lysosomal storage diseases (LSDs) have generated interest in screening newborns for these conditions. We present performance validation data on a digital microfluidic platform that performs multiplex enzymatic assays for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases. METHODS: We developed an investigational disposable digital microfluidic cartridge that uses a single dried blood spot (DBS) punch for performing a 5-plex fluorometric enzymatic assay on up to 44 DBS samples. Precision and linearity of the assays were determined by analyzing quality control DBS samples; clinical performance was determined by analyzing 600 presumed normal and known affected samples (12 for Pompe, 7 for Fabry and 10 each for Hunter, Gaucher and Hurler). RESULTS: Overall coefficient of variation (CV) values between cartridges, days, instruments, and operators ranged from 2 to 21%; linearity correlation coefficients were ≥0.98 for all assays. The multiplex enzymatic assay performed from a single DBS punch was able to discriminate presumed normal from known affected samples for 5 LSDs. CONCLUSIONS: Digital microfluidic technology shows potential for rapid, high-throughput screening for 5 LSDs in a newborn screening laboratory environment. Sample preparation to enzymatic activity on each cartridge is less than 3h.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Microfluídica/métodos , Tamizaje Neonatal , alfa-Glucosidasas/sangre , Pruebas con Sangre Seca , Pruebas de Enzimas , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Recién Nacido , Enfermedades por Almacenamiento Lisosomal/sangre , Enfermedades por Almacenamiento Lisosomal/clasificación , Microfluídica/instrumentación , Microfluídica/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , alfa-Glucosidasas/deficienciaRESUMEN
A peptide nucleic acid (PNA) targeting a splice junction of the murine PTEN primary transcript was covalently conjugated to various basic peptides. When systemically administered to healthy mice, the conjugates displayed sequence-specific alteration of PTEN mRNA splicing as well as inhibition of full length PTEN protein expression. Correlating activity with drug concentration in various tissues indicated strong tissue-dependence, with highest levels of activity observed in adipose tissue. While the presence of a peptide carrier was found to be crucial for efficient delivery to tissue, little difference was observed between the various peptides evaluated. A second PNA-conjugate targeting the murine insulin receptor primary transcript showed a similar activity profile, suggesting that short basic peptides can generally be used to effectively deliver peptide nucleic acids to adipose tissue.