A Stepwise, Prevalence-Driven Framework to Resolve Prenatal Testing Discrepancies: Integrating Institutional Expertise and a β-Thalassemia Case Study

Background: While direct testing in prenatal diagnosis provides superior specificity, incorporating indirect methods is also valuable. Concordant results between direct and indirect methods reinforce diagnostic accuracy, while discordant results must be carefully assessed, as they may indicate technical errors or reveal underlying biological mechanisms which would ultimately advance our understanding of inherited disorders. Despite their clinical and diagnostic significance, no standardized protocol currently exists for resolving such conflicts. Based on years of experience in prenatal testing, we have proposed a prevalence-driven, stepwise framework that accounts for both the frequency and significance of such discrepancies in prenatal diagnostics. We validated this protocol using a representative case of β-thalassemia, demonstrating its effectiveness in addressing complex diagnostic scenarios. Case Presentation: we used a systematic approach to identify a fetus genotype using a combination of direct and indirect molecular methods for β-thalassemia disease. The results of the direct molecular testing showed a heterozygous state while RFLP and haplotype analyses showed that the fetus is affected with β-thalassemia in homozygous state. After ruling out the possible causes including technical errors, maternal contamination, non-paternity, mosaicism in the placenta and chimerism, we concluded that the discrepancy was caused by a balanced recombination event within the β-globin gene cluster during parental meiosis. Direct testing results were ultimately deemed definitive due to their higher specificity and directness. Conclusions: This report underscores the necessity of addressing such inconsistencies systematically and proposes a systematic resolution framework to ensure diagnostic accuracy. By adopting the outlined approach, laboratories can confidently resolve such discrepancies and build confidence in the molecular testing process even in complex or rare cases.