Non-invasive fetal RHD genotyping
Editorial Commentary

Non-invasive fetal RHD genotyping

Frederik Banch Clausen^

Laboratory of Blood Genetics, Department of Clinical Immunology, Copenhagen University Hospital, Copenhagen, Denmark

^, ORCID: 0000-0003-2487-5373

Correspondence to: Frederik Banch Clausen, DMSc, PhD. Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark. Email:

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Blood. The article did not undergo external peer review.

Comment on: Londero D, Stampalija T, Bolzicco D, et al. Fetal RHD detection from circulating cell-free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA. Transfus Med 2019;29:408-14.

Received: 23 April 2020; Accepted: 21 May 2020; Published: 30 September 2020.

doi: 10.21037/aob-20-36

From analysis of a standard blood sample from a pregnant woman, it is possible to predict fetal blood groups using non-invasive prenatal testing of cell-free fetal DNA (cffDNA) (1). Knowledge of fetal blood groups is valuable for identifying incompatibility cases where the fetus has inherited a paternal gene or gene variation determining an antigen which the pregnant woman does not have herself and thus is unknown to the maternal immune system. During a pregnancy, the maternal immune system may produce antibodies against the antigen. In the form of IgG, the maternal antibodies can be transferred across the placenta into the fetal blood circulation where the antibodies can facilitate the destruction of fetal red blood cells, leading to hemolytic disease of the fetus and newborn (HDFN) (2).

For RhD negative pregnant women, fetal RHD genotyping can help clinicians in the management of D immunized women, securing diagnosis and timely treatment. For non-immunized RhD negative pregnant women, Rh prophylaxis is used to hinder the women in becoming immunized (2). Fetal RHD genotyping can guide the targeted use of Rh prophylaxis, so that only those RhD negative pregnant women who carry an RhD positive fetus are administered antenatal prophylaxis (3). This strategy adopts a rational use of often limited anti-D immunoglobulin and avoids unnecessary treatment of women carrying an RhD negative fetus (4). It is an important objective to avoid unnecessary treatment of pregnant women, thus avoiding unnecessary exposure of anti-D and the potential risk of infection, although highly theoretical, associated with the exposure to a blood-derived product.

Since the first fetal RHD genotyping assays were introduced around 20 years ago, several countries worldwide have implemented a service for D immunized women (5,6), and in several European countries, fetal RHD genotyping is now implemented as an antenatal screening assay to guide targeted prophylaxis on a national basis (7).

cffDNA is present in the maternal blood in very low concentrations, especially in early pregnancy (3). Consequently, reliable fetal RHD genotyping requires careful attention to each step of the method to ensure high assay sensitivity. In addition, the high polymorphism of the Rh blood group system requires careful attention in the selection of the RHD exons to be tested and the interpretation of the results.

In this issue, Londero et al. present a validation of an assay for fetal RHD genotyping intended for guiding targeted prophylaxis (8). Londero et al. use a well-validated RHD exon combination to amplify the fetal RHD. With samples from 133 pregnant RhD negative women, of which more than half were taken from the first trimester, they demonstrate 100% concordance between their assay-based prediction of the fetal RhD type and the newborn RhD type, assessed after birth by standard cord blood serology.

In addition to various validation steps, they also investigate the performance on frozen cffDNA which is shown to perform equally as well as testing the DNA directly after DNA extraction. Having the possibility to freeze extracted cffDNA and test it later provides valuable flexibility in a clinical setting.

They also compare manual and automated DNA extraction, implementing automated DNA extraction, which offers higher reproducibility, less hands-on time and is known to limit the risk of contamination and human error (3).

Londero et al. found one result which suggested a potential fetal RHD variant, but a correct prediction was made. It is always valuable to investigate such cases to get an understanding of the variants that exist in the population and how they may affect the outcome of the assay. However, in the routine clinical handling of cases with inconclusive results, prophylaxis can simply be offered without the need of deeper investigation into the type of variant.

Noninvasive prenatal testing of cffDNA for fetal blood grouping has become an important tool assisting in the prevention of HDFN. Fetal RHD genotyping represent one of the first successful clinical applications of cffDNA, and its performance in guiding targeted prophylaxis has been documented extensively in the literature, underlining its capacity for clinical use (7). The study from Londero et al. adds further evidence to the reliability of fetal RHD genotyping.


Funding: None.


Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at The author has no conflicts of interest to declare.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See:


  1. van der Schoot CE, Winkelhorst D, Clausen FB. Noninvasive Fetal Blood Group Typing. In: Klein HG, Page-Christiaens L. editors. Noninvasive Prenatal Testing (NIPT): Applied Genomics in Prenatal Screening and Diagnosis. London: Academic Press; 2018:125-56.
  2. de Haas M, Thurik FF, Koelewijn JM, van der Schoot CE. Haemolytic disease of the fetus and newborn. Vox Sang 2015;109:99-113. [Crossref] [PubMed]
  3. van der Schoot CE, de Haas M, Clausen FB. Genotyping to prevent Rh disease: has the time come? Curr Opin Hematol 2017;24:544-50. [Crossref] [PubMed]
  4. Kent J, Farrell AM, Soothill P. Routine administration of Anti-D: the ethical case for offering pregnant women fetal RHD genotyping and a review of policy and practice. BMC Pregnancy Childbirth 2014;14:87. [Crossref] [PubMed]
  5. Clausen FB, Damkjær MB, Dziegiel MH. Noninvasive fetal RhD genotyping. Transfus Apher Sci 2014;50:154-62. [Crossref] [PubMed]
  6. Zhu YJ, Zheng YR, Li L, et al. Diagnostic accuracy of non-invasive fetal RhD genotyping using cell-free fetal DNA: a meta analysis. J Matern Fetal Neonatal Med 2014;27:1839-44. [Crossref] [PubMed]
  7. Clausen FB. Lessons learned from the implementation of non-invasive fetal RHD screening. Expert Rev Mol Diagn 2018;18:423-31. [Crossref] [PubMed]
  8. Londero D, Stampalija T, Bolzicco D, et al. Fetal RHD detection from circulating cell-free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA. Transfus Med 2019;29:408-14. [Crossref] [PubMed]
doi: 10.21037/aob-20-36
Cite this article as: Clausen FB. Non-invasive fetal RHD genotyping. Ann Blood 2020;5:27.