BACKGROUND: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. RESULTS: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤ 6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. CONCLUSIONS: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.
BACKGROUND: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. RESULTS: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤ 6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. CONCLUSIONS: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.
Authors: Maria Alessandra Saltarelli; Rossella Ferrante; Francesca Di Marcello; Daniela David; Silvia Valentinuzzi; Lucrezia Pilenzi; Luca Federici; Claudia Rossi; Liborio Stuppia; Stefano Tumini Journal: Int J Environ Res Public Health Date: 2022-06-04 Impact factor: 4.614
Authors: Mark R de Hora; Natasha L Heather; Tejal Patel; Lauren G Bresnahan; Dianne Webster; Paul L Hofman Journal: Int J Neonatal Screen Date: 2020-01-28