21-deoxycortisol as a second-tier test in congenital adrenal hyperplasia newborn screening in The Netherlands: two-year evaluation

Objective Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an inherited adrenal steroid synthesis disorder included in Dutch newborn screening (NBS) since 2002. Screening involves measuring 17-hydroxyprogesterone (17-OHP) in dried blood spots (DBS) with gestational age-adjusted cut-offs. Since October 2021, a second-tier 21-deoxycortisol (21-DF) test has replaced the 17-OHP measurement in a second heel prick after inconclusive results. This study evaluates the performance of the second-tier test from 1 October 2021 to 30 September 2023. Design 17-OHP was measured by immunoassay in regional NBS laboratories. DBS with inconclusive and positive 17-OHP results was sent to Amsterdam UMC for 21-DF measurement by liquid chromatography tandem mass spectrometry. Genetic analysis of CYP21A2, CYP11B1, POR and HSD3B2 was performed on DBS from newborns with false-positive second-tier results. Results Over 2 years, 21-DF was measured in DBS of 147 newborns (=0.04%). Twenty newborns were directly referred to a paediatric endocrinologist based on positive 17-OHP results: 15 had a positive 21-DF and were diagnosed with classical CAH (genetically confirmed), while five were first-tier false-positives. Of 127 newborns with inconclusive 17-OHP results, three had a positive 21-DF and were referred but not diagnosed with CAH: second-tier false-positives. In total, 8/23 referred newborns were false-positives. Genetic analysis of six false-positive second-tier DBS showed pathogenic CYP21A2 variants in five. Conclusions The modified protocol improved CAH screening by preventing 127 heel pricks in 2 years and reducing unnecessary referrals (currently 2.4%, previously 7.7%). 5/6 false-positive second-tier tests were most likely due to non-classical CAH. Further optimisation of cut-offs may prevent these false-positives.