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

TLDR

Newborn screening for congenital adrenal hyperplasia (CAH) typically measures 17α‑hydroxyprogesterone (17‑OHP) by immunoassay, but this approach has high false‑positive rates. The study aimed to develop a steroid profiling method using LC‑MS/MS to simultaneously measure 17‑OHP, androstenedione, and cortisol in dried blood spots to reduce false‑positive results. The method extracts steroids from dried blood spots into diethyl ether, evaporates, dissolves in LC mobile phase, and quantifies 17‑OHP, androstenedione, and cortisol by LC‑MS/MS in selected‑reaction monitoring mode using a deuterium‑labeled internal standard. Using the LC‑MS/MS profiling, 86 of 101 false‑positive samples were reclassified as normal, and all 742 normal and 14 true‑positive samples were correctly identified, confirming that steroid profiling reduces false positives in newborn CAH screening.

Abstract

Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-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.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 d(8)-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.Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 microg/L. At a cutoff for 17-OHP of 12.5 microg/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.Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.