Second-tier testing for 21-hydroxylase deficiency in the Netherlands; a newborn screening pilot study.

Background Newborn screening (NBS) for classic congenital adrenal hyperplasia (CAH) consists of 17-hydroxyprogesterone (17-OHP) measurement with gestational age-adjusted cut-off values. A second heel puncture (HP) is performed in newborns with inconclusive results to reduce false-positives. We assessed the accuracy and turnaround time of the current CAH NBS-algorithm and compared this to alternative algorithms by performing a second-tier 21-deoxycortisol (21-DF) pilot-study. Methods Dried blood spots (DBS) of newborns with inconclusive and positive 17-OHP (immunoassay) first HP results were sent from regional NBS laboratories to the Amsterdam UMC Endocrine Laboratory. In 2017-2019 21-DF concentrations were analyzed by liquid chromatography-tandem mass spectrometry, in parallel with routine NBS. Diagnoses were confirmed by mutation analysis. Results 328 DBS were analyzed. 37 newborns had confirmed classic CAH, 33 were false-positive and 258 were categorized as negative in the second HP following the current algorithm. In the second-tier, all 37 confirmed CAH had elevated 21-DF concentrations, all 33 false-positives and 253/258 second HP negatives had undetectable 21-DF concentrations. The elevated 21-DF of the other five newborns may be NBS false-negatives or second-tier false-positives. Adding the second-tier to inconclusive first HPs reduced the number of false-positives to 11 and prevented all 286 second HPs. Adding the second-tier to both positive and inconclusive first HPs eliminated all false-positives at the cost of delayed referral for 31 CAH patients (1-4 days). Conclusion Application of the second-tier 21-DF measurement to inconclusive first HPs improved our CAH NBS by reducing false-positives, abolishing the second HP and thereby shortening referral time.