RH alleles: historical milestones, molecular advances, and clinical application

The Rh blood group system was first described in 1939 when Levine and colleagues reported a case involving a woman (group O) who, after delivering a stillborn child with erythroblastosis fetalis, experienced a severe transfusion reaction when transfused with blood from her husband (group O). Her serum agglutinated red blood cells (RBCs) from 80% of Caucasian ABO-compatible donors (1). In the following year, Landsteiner and Wiener (2) immunized rabbits with RBCs from Macaca mulatta (Macacus rhesus in the original paper), and their sera agglutinated 85% of human RBC samples. Initially, it was thought that the animal and human antibodies recognized a common factor, Rh, on the surface of rhesus and human RBCs. It was soon realized that this was not the case, but the terms Rh factor and anti-Rh continued to be used. Over the years, researchers began to recognize the complexity of the Rh system. In the 1940s, the major Rh antigens were recognized, and in 1946, Stratton (3) reported that some D-positive RBCs showed variable agglutination with certain anti-D sera, while others completely failed to agglutinate the cells. This phenotype was initially designated as D^U. Subsequently, investigations of D positive individuals with alloanti-D revealed that they were lacking or had altered D epitopes; such phenotype was classified as partial-D (4,5). Originally, D variants were defined using a panel of anti-D (4). However, the description of the molecular basis of the RH system in the 90’s along with molecular advances, led to the discovery of several novel variants and alleles. This series provides the historical evolution of the discovery of novel RH alleles, recent technological developments, challenges in characterizing novel alleles, and defining their clinical impact. The articles were written by international experts on genomics of blood groups.

In the first article, Dr. Cotorruelo (6) provides an introductory overview of the molecular basis of the Rh system and the new RH allele discoveries over time. He discusses the contribution of the molecular study of the Rh system to transfusion and obstetric medicine fields such as the fetal RHD genotyping to identify the fetus who is not at risk of HDN, RH variants genotyping for chronically transfused patients and as a tool for guiding anti-D prophylaxis, and molecular matching for sickle cell disease patients.

In the second article, Dr. Castilho (7) addresses the complexity of RH variants, demonstrating that defining the clinical impact of a variant extends beyond its genetic basis. The interaction of proteins and the trimeric conformation of Rh proteins can influence the risk of alloimmunization and the clinical significance of antibodies. She discusses future directions, including the incorporation of 3D analysis and computational tools to predict the functional effect of novel RH alleles. She describes an interesting case that illustrates how the 3D analysis could be used when new RH variants are detected to get insights into the clinical phenotype that may be predicted from genotyping.

In the third article, Dr. Madgett and colleagues (8) provide an overview of the historical facts leading to the determination of the molecular basis for the RH gene. The authors report the evolution of molecular techniques arriving at next generation sequencing (NGS) for RH genotyping. The authors show how their NGS RHD protocol has been successfully used to identify novel RHD variants and discuss the current challenges when using NGS for RH genotyping. This review underscores the substantial advantage of NGS in identifying novel variants which is not possible using microarray/bead technology.

In the fourth article, Dr. Keller (9) shares valuable insights from managing a national reference laboratory of molecular immunohematology for over a decade that will help readers select genotyping assays and interpret results accurately. Dr. Keller provides a comprehensive overview of the challenges and limitations of commercially available red cell genotyping panels and the risks and impact of misinterpreting RH alleles. Additionally, she documents the complexity of the RH system, which can disrupt accurate and safe RH classification. Her narrative was supported by laboratory cases, making it easy for readers to visualize and relate to them in their daily laboratory operations.

As Guest Editors, we are grateful to the expert authors for agreeing to contribute, dedicating their time to writing, and for sharing their experiences that bring inestimable value to this series. We hope that this special series on ‘Novel RH alleles’ can emphasize the importance of accurately detecting novel alleles and their consequences for transfusion practice.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Blood for the series “Novel RH Alleles”. The article did not undergo external peer review.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-2024-01/coif). The series “Novel RH Alleles” was commissioned by the editorial office without any funding or sponsorship. C.P.A., E.S., and M.R. served as the unpaid Guest Editors of the series. E.S. and M.R. claim that these comments are an informal communication and represent their own best judgement. These comments do not bind or obligate FDA. The authors have no other conflicts of interest to declare.

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: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

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2. Landsteiner K, Wiener AS. An Agglutinable Factor in Human Blood Recognized by Immune Sera for Rhesus Blood. Proc Soc Exp Biol Med 1940;43:223. [Crossref]
3. Stratton F. A new Rh allelomorph. Nature 1946;158:25. [Crossref] [PubMed]
4. Tippett P, Sanger R. Observations on subdivisions of the Rh antigen D. Vox Sang 1962;7:9-13. [Crossref] [PubMed]
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6. Cotorruelo C. Elucidation of the molecular bases of the Rh system and its contribution to transfusion and obstetric medicine—historical and current perspective: a review. Ann Blood 2023;8:37. [Crossref]
7. Castilho L. Implication of novel RH alleles in blood transfusion therapy. Ann Blood 2023;8:38. [Crossref]
8. Madgett TE, Tounsi WA, Halawani AJ, et al. RHD molecular analysis—from discovery to next generation sequencing. Ann Blood 2023;8:36. [Crossref]
9. Keller MA. Challenges with assigning RH alleles and accurately predicting phenotypes using commercially-available genotyping kits: a narrative review. Ann Blood 2024;9:4. [Crossref]