Prenatal RHD genotyping in Croatia: preliminary results

Objectives: Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed.

Materials and methods: Evaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12-36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and β-actin genes digested with BsTUI were used.

Results: We identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively.

Conclusion: Automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.