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Newborn Screening for Congenital Adrenal Hyperplasia: Additional Steroid Profile using Liquid Chromatography-Tandem Mass Spectrometry

Screening-Labor Hannover, D-30430 Hannover, Germany

Address all correspondence and requests for reprints to: Dr. Nils Janzen, Screening-Labor Hannover, Am Steinweg 13B, D-30952 Ronnenberg, Germany. E-mail: n.janzen{at}metabscreen.de.

Background: Neonatal screening programs for congenital adrenal hyperplasia (21-CAH) using an immunoassay for 17-hydroxyprogesterone (17-OHP) generate a high rate of positive results attributable to physiological reasons and to cross-reactions with steroids other than 17-OHP, especially in preterm neonates and in critically ill newborns.

Methods: To increase the specificity of the screening process, we applied a liquid chromatography-tandem mass spectrometry method quantifying 17-OHP, 11-deoxycortisol, 21-deoxycortisol, cortisol, and androstenedione. The steroids were eluted in aqueous solution containing d₈-17-OHP and d₂-cortisol and quantified in multiple reaction mode.

Results: Detection limit was below 1 nmol/liter, and recovery ranged from 64% (androstenedione) to 83% (cortisol). Linearity was proven within a range of 5–100 nmol/liter (cortisol, 12.5–200 nmol/liter), and total run time was 6 min. Retrospective analysis of 6151 blood samples and 50 blood samples from newborns with clinically confirmed 21-CAH, as well as prospective analysis of 1609 samples of a total of 242,500 testing positive in our routine 17-OHP immunoassay, allowed clear distinction of affected and nonaffected newborns. High levels of 21-deoxycortisol were only found in children with 21-hydroxylase deficiency. Calculating the ratio of 17-OHP to 21-deoxycortisol divided by cortisol further increased the sensitivity of the method.

Conclusion: Our liquid chromatography-tandem mass spectrometry procedure as a second-tier test can be used to reduce false-positive results of standard 21-CAH screening. The short total run time of 6 min allows for immediate reanalysis of all immunoassay results above the cutoff.

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