Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis
Letter to the Editor

Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis

Donatella Londero, Vincenzo De Angelis

Department of Transfusion Medicine, ASUFC-Udine, Italy

Correspondence to: Vincenzo De Angelis. Department of Transfusion Medicine, Azienda Sanitaria Universitaria Friuli Centrale, Piazzale S. Maria della Misericordia, 15- 33100 Udine, Italy. Email:

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

Response to: Clausen FB. Non-invasive fetal RHD genotyping. Ann Blood 2020. doi: 10.21037/aob-20-36

Received: 24 June 2020; Accepted: 12 July 2020; Published: 30 September 2020.

doi: 10.21037/aob-20-43

In his commentary on “Non-invasive fetal RHD-genotyping”, Frederik Banch Clausen referred to our recently published article (1). We agree on the contents and we would like to provide some additional information on our study and on its implementation.

Fetal RHD genotyping on cell-free fetal DNA (cf-DNA) from RhD-negative women can be used to guide targeted antenatal prophylaxis for the prevention of RhD immunization: the knowledge of fetal RhD type in fact can direct and restrict the use of prenatal anti-D Ig exclusively to RhD negative women carrying an RhD positive fetus (60% of individuals of European descent). This approach optimizes the use of a plasma-derived medicinal product, which is becoming more and more a limited resource (2-4), also avoiding any unnecessary exposition to plasma-derived medicinal products.

Besides the mandatory validation steps, the implementation of any diagnostic test requires however to adapt the organizational approaches. In our study, in addition to standard validation parameters (analytical sensitivity, lower limit of detection, analytical specificity, assay precision and diagnostic accuracy) we investigated the performance of the diagnostic kit to optimize routine laboratory organization, by assessing the feasibility of automatic DNA extraction, to improve efficiency and standardization of the cf-DNA extraction phase, and the reliability of preserving extracted cf-DNA in frozen state before real-time quantitative PCR assays.

Validation results were very satisfying for all quality parameters and added evidence to the reliability of a large-scale fetal RHD genotyping in clinical setting; in particular, it has been achieved an excellent concordance between the fetal RHD genotype predicted by the assay on different gestational week samples and the RhD phenotype assessed after birth by standard cord blood serology, ensuring an evidence-based use of antenatal anti-D Ig prophylaxis in pregnant women at risk of HDFN (Table 1).

Table 1
Table 1 Diagnostic accuracy in relation to gestational week
Full table

These results were crucial for planning and implementing the first Italian region-wide screening service for fetal RHD genotyping at 22th–24th gestational weeks (gw), taking also as a model the North-European countries experience (5-8). We have demonstrated that it is feasible to obtain the predicted fetal RhD phenotype before the 28th gw (when ante-natal prophylaxis is usually administered in Italy) in all RhD-negative pregnant women previously typed before the 12th gw; therefore, the antenatal screening program has been effectively implemented, by collecting, storing and automatically extracting DNA from maternal plasma to analyze fetal RHD gene (exons 5, 7 and 10). In our Region, tests are provided free of charge by the Regional Health System from November 2019 and they have received excellent user satisfaction. The adoption of fetal RHD antenatal testing is confirmed to be highly reliable and the centralization of the text in a single laboratory guarantees the quality of the results, the concordance of reports and the sustainability of the costs, representing an excellent guide to targeted use of prophylaxis.


Funding: None.


Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at DL reports grants from Friuli Venezia Giulia Region, during the conduct of the study. VDA reports grants from Friuli Venezia Giulia Region, during the conduct of the study.

Ethical Statement: The authors are 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:


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doi: 10.21037/aob-20-43
Cite this article as: Londero D, De Angelis V. Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis. Ann Blood 2020;5:29.