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Improved Specificity of Newborn Screening for Congenital Adrenal Hyperplasia by Second-Tier Steroid Profiling Using Tandem Mass Spectrometry

Departments of¹ Laboratory Medicine and Pathology, ³ Medical Genetics, ⁴ Pediatric and Adolescent Medicine, and ⁵ Biostatistics, Mayo Clinic College of Medicine, Rochester, MN. ² Department of Pediatrics, John Stroger Jr. Hospital of Cook County, Chicago, IL. ⁶ Applied Biosystems, Monza, Italy. ⁷ Minnesota Department of Health, Minneapolis, MN.

^aAddress correspondence to this author at: Biochemical Genetics Laboratory-Hilton 330, Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. Fax 507-266-2888; e-mail matern{at}mayo.edu.

Background: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography–tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots.

Methods: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d₈-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening.

Results: Intra- and interassay CVs for 17-OHP were 7.2–20% and 3.9–18%, respectively, at concentrations of 2, 30, and 50 µg/L. At a cutoff for 17-OHP of 12.5 µg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified.

Conclusion: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.

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