A real-time PCR-based noninvasive prenatal RHD screening assay optimized for population-specific RHD allelic spectra in China.

BACKGROUND: Prenatal RhD screening enables risk stratification for RhD-associated hemolytic disease of the fetus and newborn (HDFN) and supports rational，evidence-based use of prophylactic anti-RhD immunoglobulin. However, noninvasive prenatal RhD screening remains challenging in the Chinese population, primarily due to population-specific RHD allelic diversity and the limited adaptability of current assay designs. METHODS: We developed and optimized a real-time PCR-based assay for noninvasive prenatal RHD genotyping, specifically designed to accommodate the spectrum of clinically relevant RHD alleles in the Chinese population. By simultaneously targeting RHD exons 5, 9, and 10, the assay enables detection of paternally inherited RHD alleles within a single analytical workflow. This assay facilitates accurate fetal RHD status determination in pregnancies involving the predominant RhD-negative alleles, including RHD*01N.01, RHD*01N.03, and RHD*01EL.01. RESULTS: The assay accurately predicted fetal RHD status across all simulated maternal genotypes, including those carrying non-deletion alleles such as RHD*01N.03 and RHD*01EL.01. CONCLUSION: This study presents a methodological framework for noninvasive prenatal RHD genotyping, specifically optimized for Chinese and other East Asian populations. Assay performance relies on distinct amplification patterns of population-specific RHD alleles across exons 5, 9, and 10. In addition, inclusion of exon 5 allows detection of RHD variants frequently observed in African populations, suggesting potential applicability across genetically diverse populations, pending further population-specific validation.