Advancing prenatal diagnosis: a comparative study of ddPCR and qPCR for non-invasive RHD genotyping in D-negative pregnancies

TRUCCO BOGGIONE, CAROLINA; MATTALONI, STELLA MARIS; AVILÉS, SOCRATES; POSNER, VICTORIA; STETTLER, SILVINA; PRINCIPI, CINTIA; PERÓN, ANA CLAUDIA; BUFFETTI, ROSANA; TORRES, OSCAR; SESMA, JULIANA; COTORRUELO, CARLOS

Congreso; 38th ISBT International Congress; 2024

ISBT

Background: Hemolytic Disease of the Foetus and Newborn (HDFN) caused by anti-D antibodies is a significant concern globally. To prevent this condition, D-negative pregnant women are routinely administered prenatal anti-D immunoprophylaxis, followed by an additional dose post-delivery of a D-positive newborn. Non-invasive fetal RHD genotyping allows an early risk assessment of this disease in pregnancies with anti-D alloantibodies and has the potential to avoid antenatal anti-D prophylaxis in non-sensitized D negative pregnant women carrying RHD negative foetuses. This approach requires sensitive methodologies capable of detecting cell-free fetal DNA (cffDNA) at early gestational ages.Aim: this study aims to evaluate a novel Droplet Digital PCR (ddPCR) technique, developed in our laboratory, against the already validated Real-Time PCR (qPCR) method for prenatal RHD genotyping in D-negative pregnant women.Samples and Methods: peripheral blood samples (10 ml) from 27 D-negative pregnant women (gestation weeks 12-38) were collected for prenatal RHD genotyping. Plasma aliquots (4 ml) were processed to extract total cell-free DNA using the QIAamp MinElute ccfDNA Midi Kit (Qiagen). The ddPCR assays were performed on a QX200 Droplet Digital PCR System (Bio-Rad), following the manufacturer's guidelines, while the Real-Time PCR reactions were run on a Step One Applied Biosystem thermocycler. In both strategies, two different regions of the RHD gene (exon 5 and exon 7) were analysed alongside a reference gene (GAPDH) and a sex-determining gene (SRY).Results: the ddPCR technique successfully genotyped all 27 cffDNA samples, identifying 3 RHD-negative and 24 RHD-positive foetuses, including 11 males and 16 females. Conversely, the Real-Time PCR method failed to characterize three samples, likely due to a low cffDNA concentration as these samples were from early gestations of 11 (n=2) and 12 (n=1) weeks. The genotype and sex determinations for the remaining 24 cffDNA samples were consistent across both ddPCR and qPCR methods. Serological analysis of cord blood samples confirmed the molecular findings with 100% accuracy.Conclusions: the ddPCR technique formulated in our laboratory proves to be a dependable and effective approach for accurately determining prenatal RHD genotype in all analyzed cffDNA samples. Given that cffDNA constitutes only 5-15% of total cfDNA, a significant advantage of ddPCR over Real-Time PCR is its superior sensitivity, particularly beneficial for samples with a low fetal cfDNA proportion.