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

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 50; no. 3; pp. 621 - 625
• Main Authors: Lacey, Jean M, Minutti, Carla Z, Magera, Mark J, Tauscher, Angela L, Casetta, Bruno, McCann, Mark, Lymp, James, Hahn, Si Houn, Rinaldo, Piero, Matern, Dietrich
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01-03-2004; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Adrenal Hyperplasia, Congenital - blood; Adrenal Hyperplasia, Congenital - diagnosis; Analytical, structural and metabolic biochemistry; Androstenedione - blood; Biological and medical sciences; Blood; Chromatography, Liquid; Deuterium; False Positive Reactions; Fundamental and applied biological sciences. Psychology; Humans; Hydrocortisone - blood; Immunoassay; Infant, Newborn; Investigative techniques, diagnostic techniques (general aspects); Liquid chromatography; Mass Screening - methods; Mass spectrometry; Medical sciences; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Progesterone - analogs & derivatives; Progesterone - blood; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Steroids; Steroids - blood; Endocrinopathy; Human; Steroid; Adrenal cortex diseases; Hyperadrenocorticism; Biochemistry; Medical screening; Enzymopathy; Congenital adrenal hyperplasia syndrome; Specificity; Newborn; Mass spectrometry MS/MS; Adrenal gland diseases; Clinical biology
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2003.027193

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.