Original article

Vol. 21 No. 6 (2023): Blood Transfusion 6-2023 (November-December)

Evaluation of an automated platform for non-invasive single-exon fetal RHD genotyping early in pregnancy


Key words: cffDNA, non-invasive fetal RHD genotyping, hemolytic disease of the newborn
Publication Date: 2023-02-14


Background - RhD immunization is still the major cause of hemolytic disease of the fetus and newborn. Fetal RHD genotyping during pregnancy followed by tailored anti-D prophylaxis for pregnant RhD-negative women carrying an RHD-positive fetus to prevent RhD immunization is a well-established practice in many countries. This study aimed to validate a platform for
high-throughput, non-invasive, single-exon, fetal RHD genotyping consisting of automated DNA extraction and PCR set-up, and a novel system for electronic data transfer to the real-time PCR instrument. We also investigated the effect of storage conditions of fresh or frozen samples on the outcome of the assay.
Materials and methods - Blood samples from 261 RhD-negative pregnant women collected in Gothenburg, Sweden, between November 2018 and April 2020 during gestation week 10-14 were either tested as fresh after storage for
0-7 days at room temperature or as thawed plasma samples previously separated and stored for up to 13 months at −80°C. Extraction of cell-free fetal DNA and PCR set-up were performed in a closed automated system. Fetal RHD genotyping was determined by real-time PCR amplification of the RHD gene exon 4.
Results - The outcome of RHD genotyping was compared with either the results obtained with serological RhD typing of newborns or with the results of RHD genotyping performed by other laboratories. No difference was observed in genotyping results when using fresh or frozen plasma during short- and long-term storage, revealing high stability of cell-free fetal DNA. The assay has shown high sensitivity (99.37%), specificity (100%), and accuracy (99.62%).
Discussion - These data confirm that the proposed platform for non-invasive, single-exon, RHD genotyping early in pregnancy is accurate and robust. Importantly, we demonstrated the stability of cell-free fetal DNA in fresh and frozen samples after short- and long-term storage.



Pauline Isakson - Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden

Cecilia Pardi - Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden

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