Prevention of anti-D prophylaxis in pregnant RhD negative females using non-invasive foetal RhD genotyping in Malta

Abstract

The discovery of cell-free foetal DNA (cffDNA) in maternal peripheral blood has revolutionised the field of non-invasive prenatal testing (NIPT), eliminating the need for invasive procedures to obtain a foetal sample. In recent years, the routine antenatal anti-D prophylaxis (RAADP) for all RhD negative women has been subject to ethical considerations. This has led to the concept of targeted RAADP, where women are treated with anti-D prophylaxis only if the foetus is determined to be RhD positive through foetal RHD genotyping. The aim of this study is to introduce NIPT foetal RHD genotyping in Malta with the goal of reducing prophylactic anti-D usage by 42%. To achieve this, the study sets out three specific objectives: 1. Design and validate a highly sensitive and specific RHD genotyping test suitable for the Maltese population; 2. Compare the effectiveness of two PCR platforms - realtime PCR (qPCR) and droplet digital PCR (ddPCR); 3. Confirm the PCR results by comparing them with the current gold standard test - the Cord Blood test. The methodology of the study was divided into five key parts: 1. Recruitment and collection of maternal and control samples; 2. Processing, extraction, and quantification of cell-free DNA; 3. Validation and analysis of qPCR and ddPCR; 4. Confirmation of both PCR platforms with the current gold standard test; 5. Analysis of results and comparison of the two PCR platforms. The successful validation of both qPCR and ddPCR demonstrated a remarkable 98.15% diagnostic accuracy. Furthermore, qPCR exhibited superior specificity, while ddPCR showed higher sensitivity. These results align with findings from studies conducted in other countries, thereby supporting the use of both PCR platforms and the introduction of NIPT in Malta. In conclusion, the implementation of NIPT foetal RHD genotyping in Malta holds promising potential for reducing the reliance on prophylactic anti-D treatment. The validation and comparison of qPCR and ddPCR have shown that both methods are highly accurate and reliable. This study contributes valuable insights to the field of prenatal testing and lays the foundation for the adoption of NIPT in Malta.