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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.

Screening newborns for inherited disorders provides an opportunity for pre-symptomatic identification and early intervention to prevent or mitigate morbidity and mortality associated with these conditions. Since the introduction of newborn screening in 1962 to screen for phenylketonuria, technological advances have enabled the screening panel to expand substantially so that it now includes more than 50 disorders. Newborn screening will continue to evolve,, and deployment of improved methodologies and incorporation of additional disorders are expected. This article provides an overview of the current state of newborn screening, and describes the disorders detectable, the methodologies employed, and the challenges involved in analyses of specimens obtained from newborns.

Classic CAH (salt-wasting and simple virilizing) meets all of the recommended criteria for newborn screening. There are reliable and efficient newborn screening tests, the disorder results in high morbidity and mortality if left undetected, there is effective treatment that reduces negative outcomes, and there is a relatively high incidence. When compared with the case findings without the benefit of screening, the data from screening programs show reduced adrenal crises, reduced incorrect sex assignments, and reduced deaths. Racial/ethnic prevalence differences are present in newborn screening program data. The Texas data indicate a lower disease frequency in African-Americans when compared with Caucasians, and international data indicate higher frequencies in native Yupik Eskimos, Brazilians, residents of La Reunion, and Filipinos. When worldwide clinical ascertainment data are compared with newborn screenng data, it is clear that newborns with CAH (especially males) die when screening is not done. To be effective in reducing mortality, newborn screening must be performed soon after birth, and the results must be available quickly so that early salt-wasting crises can be averted. It is preferable that newborn screening laboratiories be operational 7 days a week, and that sample delivery from the collection site to testing laboratory be as efficient as possible, including weekends and holidays, so that undue testing delays are not encountered. These two requirements pose major challenges for most programs, but they are critical to optimal screening outcome. Based on the studies in Texas, with second screening samples collected at approximately 2 weeks of age, some newborns with simple virilizing CAH are missed on initial screening using current testing protocols. There is need to set a screening cut-off such that the false-positive rate does not oversaturate the follow-up system, in part owing to the insensitivity of current kit methodologies and the biochemical manifestations of CAH. With advances in genetic testing procedures and improved automation techniques, it may soon be possible for CAH screening programs to include genotyping as a second-tier confirmation as a part of the newborn screening protocol. Despite the fact that CAH is a continuum of disorders, the correlation between genotype and phenotype is fairly consistent in most cases. For the purpose of screening, genotyping will likely be useful only for differential diagnoses of non-salt wasters, given the necessary time constraints and expense of obtaining genotypes and the need for immediate diagnosis/treatment of salt wasters. It is hoped that newborn screening programs will begin to provide answers to some of these question in addition to their primary function of reducing the morbidity and mortality resulting from CAH.

A pilot newborn screening program for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) was conducted in Alaska using a 3-mm disc filter paper elution technique of capillary whole blood for 17-hydroxyprogesterone (17-OHP) by RIA. The filter paper specimens were provided via the mandated newborn screening program. The highest values of 17-OHP in a control population of 4569 consecutive normal neonates, aged 2–14 days, was 40 pg/disc, and the range of values for 20 newborns with proven CAH was 57–980 pg/disc. All Alaskan newborns with 17-OHP levels of 57 pg/disc or greater were referred for diagnostic workup, and initially, those with 17-OHP levels of 41–56 pg/disc were recalled for a second specimen. However, the range for recall was changed subsequently to the 17-OHP level greater than 51 pg/disc, since no newborns with 17-OHP level below 50 pg/disc were found to be affected in the initial screening. In a 30-month period, the Alaskan newborns screened on the third day of life consisted of a t...

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.

An enzyme immunoassay of 21-deoxycortisol (21-DOF) in plasma and dried blood spotted on filter paper has been developed. 21-DOF was conjugated to horseradish peroxidase by the mixed anhydride method. Separation of free and bound fractions was done by the use of insolubilized antibody, prepared by coating polyacetal beads with purified IgG of goat anti-rabbit IgG serum. The enzyme activity was measured by the fluorophotometric method using 3-(p-hydroxyphenyl)propionic acid and H2O2 as substrates. The sensitivity of the present method was 0.5 pg/tube for 21-DOF. The intra- and interassay coefficients of variation were 3.2 and 8.2, and 7.9 and 9.2% respectively. The present enzyme immunoassay could be applied to mass-screening for congenital adrenal hyperplasia.