Concurrent Confirmation and Differential Diagnosis of Congenital Adrenal Hyperplasia from Dried Blood Spots: Application of a Second-Tier LC-MS/MS Assay in a Cross-Border Cooperation for Newborn Screening

Abstract

Background/Aims: Newborn screening for congenital adrenal hyperplasia (CAH) is generally performed using 17- hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA®). The primary screening results must be confirmed due to high false-positive rates; however, the need to obtain a separate specimen can hamper early recognition, differential diagnosis and treatment. We aimed to develop a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that allows both the confirmation and differential diagnosis of CAH using the same dried blood spot (DBS) as in primary screening. Methods: An LC-MS/MS assay for cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone was developed, validated and applied to a total of 163 DBS samples tested positive in primary newborn screening in a cross-border cooperation. Results: Excellent baseline resolution and reliable determination of all analytes were achieved in DBS samples following simple sample preparation without derivatization. Of a total of 163 DBS samples tested positive in primary screening, the 21-hydroxylase-deficient form of CAH was confirmed in 1 sample. Conclusions: The present LC-MS/MS assay was successfully applied as a second-tier test in a cross-border cooperation for newborn screening. The assay allows concurrent confirmation and differential diagnosis of CAH and can be performed on the same DBS samples as in primary screening, enabling early diagnosis and treatment.

Newborn Screening Protocols and Positive Predictive Value for Congenital Adrenal Hyperplasia Vary across the United States

“…Implementation of a second screen at two weeks of life improves the positive predictive value as noted but adds cost and may be impractical or logistically difficult in some regions. A second tier test, specifically liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been cited as a useful adjunctive test [ 22 , 23 ] that may be adapted to measure an extended steroid profile [ 24 ], although it is not currently suitable for high-volume screening at the first tier due to time constraints. At present, genotyping CYP21A2 is not utilized in most US programs for either primary or secondary screening due to the complexity of test interpretation, time constraints, and costs [ 2 ], although this may change in the future.…”

Evaluation of the Dutch neonatal screening for congenital adrenal hyperplasia

“…Additional measurement of 21-deoxycortisol (21 DF) has been reported as an effective method to decrease false positive screening results 30–32. 21 DF is the 11-hydroxylated product of 17-OHP.…”

“…In unaffected neonates, 21 DF is barely detectable. The additional measurement of 21 DF is therefore a promising method to reduce false positive screening results 30–32. Furthermore, LC-MS/MS takes quite some time before results are known.…”

Congenital Adrenal Hyperplasia: clinical symptoms and diagnostic methods

“…On the other hand, glucocorticoid therapy during pregnancy can result in false negative results (King et al, 2001;Gatelais et al, 2004). A positive result should be confirmed by a second, more advanced analytical method such as high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) or gas chromatography mass spectrometry (GC-MS) (Shackleton, 1986;Monostori et al, 2015). Chromatographic methods allow simultaneous measurement of several analytes in the sample and determination of the precursor/product ratio (e.g.…”