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Received on September 15, 2003
Accepted on November 11, 2003
Endocrinology and Metabolism |
1 Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN
2 Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN and Department of Pediatrics, John Stroger Jr. Hospital of Cook County, Chicago, IL
3 Applied Biosystems, Monza, Italy
4 Minnesota Department of Health, Minneapolis, MN
5 Department of Biostatistics, Mayo Clinic College of Medicine, Rochester, MN
6 Departments of Laboratory Medicine and Pathology, Medical Genetics, and Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, MN
* To whom correspondence should be addressed. 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.© 2004 American Association for Clinical Chemistry
The following articles in journals at HighWire Press have cited this article:
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  D. J. Dietzen, P. Rinaldo, R. J. Whitley, W. J. Rhead, W. H. Hannon, U. C. Garg, S. F. Lo, and M. J. Bennett National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Follow-Up Testing for Metabolic Disease Identified by Expanded Newborn Screening Using Tandem Mass Spectrometry; Executive Summary Clin. Chem., September 1, 2009; 55(9): 1615 - 1626. [Abstract] [Full Text] [PDF]
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S. J. Soldin and O. P. Soldin Steroid Hormone Analysis by Tandem Mass Spectrometry Clin. Chem., June 1, 2009; 55(6): 1061 - 1066. [Abstract] [Full Text] [PDF]
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 N. Janzen, M. Peter, S. Sander, U. Steuerwald, M. Terhardt, U. Holtkamp, and J. Sander Newborn Screening for Congenital Adrenal Hyperplasia: Additional Steroid Profile using Liquid Chromatography-Tandem Mass Spectrometry J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2581 - 2589. [Abstract] [Full Text] [PDF]
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M. M. Kushnir, A. L. Rockwood, W. L. Roberts, E. G. Pattison, W. E. Owen, A. M. Bunker, and A. W. Meikle Development and Performance Evaluation of a Tandem Mass Spectrometry Assay for 4 Adrenal Steroids Clin. Chem., August 1, 2006; 52(8): 1559 - 1567. [Abstract] [Full Text] [PDF]
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S. Kosel, S. Burggraf, R. Fingerhut, H. G. Dorr, A. A. Roscher, and B. Olgemoller Rapid Second-Tier Molecular Genetic Analysis for Congenital Adrenal Hyperplasia Attributable to Steroid 21-Hydroxylase Deficiency Clin. Chem., February 1, 2005; 51(2): 298 - 304. [Abstract] [Full Text] [PDF]
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C. Z. Minutti, J. M. Lacey, M. J. Magera, S. H. Hahn, M. McCann, A. Schulze, D. Cheillan, C. Dorche, D. H. Chace, J. F. Lymp, et al. Steroid Profiling by Tandem Mass Spectrometry Improves the Positive Predictive Value of Newborn Screening for Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 3687 - 3693. [Abstract] [Full Text] [PDF]
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D. Marsden and C. A. Larson Emerging Role for Tandem Mass Spectrometry in Detecting Congenital Adrenal Hyperplasia Clin. Chem., March 1, 2004; 50(3): 467 - 468. [Full Text] [PDF]
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