Enhancing primary newborn screening efficiency for congenital adrenal hyperplasia with LC-MS/MS/MS

Newborn screening (NBS) for congenital adrenal hyperplasia (CAH) by immunoassay (IA) suffers from analytical limitations like cross reactivity and matrix effects. Alternative tandem-mass spectrometric approaches are not suitable for high-throughput screening due to long analysis times. In this study, a rapid 1.5 min LC-MS/MS/MS (MS³) assay was applied for the first time, maturity-dependent cutoffs were determined, and the diagnostic efficiency was compared to IA for primary CAH NBS.

1730 selected residual dried blood samples from routine NBS (gestation week 24-41) were subjected to online-SPE-LC-MS³ quantitation of 17-Hydroxyprogesterone (17-OHP). This approach utilizes two-stage fragmentation (MS³, 17-OHP m/z 329/285/123) carried out by triple quadrupole-ion trap hybrid MS. Cutoff values were calculated depending on gestational age (GA) and birthweight (BW). Diagnostic efficiency was compared to an established IA for primary CAH screening.

Intermediate precision was 10-13 % with an accuracy of 99-101 % (n = 50). Cutoff values based on GA as well as BW decreased with increasing maturity and weight of the newborns, ranging from 136 nmol/L whole blood for extreme preterm babies to 15 nmol/L whole blood for term babies. Combining LC-MS³ with GA- and BW-dependent cutoffs reduced the number of false-positive results from 731 to 15, while identifying all confirmed cases of CAH.

By application of LC-MS³ and the combinatory cutoff approach, the positive predictive value for CAH-screening improved from 1% to 32%, presenting a major benefit in diagnostic efficiency over the commonly applied IA strategy. This underlines the potential of MS³ technology in clinical use.