After 70 years, serological RhD determination remains a challenge: DNA to the rescue

Abstract

After ABO system antigens, RhD is the most clinically significant blood group antigen. This is reflected in its high immunogenicity and potential to cause haemolytic transfusion reactions (HTR) and severe haemolytic disease of the newborn (HDN). Thus, correct determination of the RhD antigen is essential for a safe transfusion strategy and adequate indications of anti‐D immunoglobulin prophylactic administration. The RhD determination challenge started in 1939 with a case history of fatal HDN and HTR in a mother who was transfused with her husband′s blood. Subsequent findings of an antibody reacting with 80% ABO compatible red blood cells (RBCs) in the serum of the afflicted woman lead to the hypothesis that the mother was lacking an antigen present on the father's and fetal RBCs and that her production of a corresponding antibody was responsible for both HDN and HTR. This hypothesis was proven to be correct. The following year, the effort to determine the origin of a causal antigen coincided with an animal immunization experiment where a similarly reacting antibody developed by injecting Rhesus monkey RBCs into rabbits and guinea‐pigs, as well as with another publication of HTRs after ABO compatible blood transfusions in patients with antibodies of apparently identical specificity. This first challenge in the ‘pre‐DNA’ era was slightly erroneously named in humans as the anti‐Rhesus antibody, and 20 years later, the animal antibody was designated anti‐LW. Meanwhile, the complexity of Rh group of antigens increased and several genetic models were suggested to explain it: Rh‐Hr single gene, three genes C, D and E and finally a two locus model (which a few years later was shown by molecular analysis to be the accurate one). At the beginning, serological determination of RhD was performed using human anti‐D, first by direct agglutination (IgM), later also using the Coombs’ test (IgG). Weak D antigens were a challenge at the time: the term ‘DU’ (now obsolete) was used for those antigens negative in direct agglutination and positive in the Coombs’ test. Weakening of the D antigen could be caused indirectly by the ‘position effect C in Trans’ or directly (mutations in the RHD gene, usually point mutations coding for amino acids in transmembranous and intracellular parts of the polypeptide). Extremely weakened D antigens are called Del, and these are serologically detected only by adsorption/elution tests (multiple genetic mechanisms). The next challenge came in the 1960s, with the observation of qualitative D variants – partial D antigens. The first cases were detected after the development of allo‐anti‐D in D‐positive individuals – immunized against that part of the D protein missing in their D epitope mosaic. Mutual reactivity of anti‐D from these individuals with different partial D antigens established the basis for their classification. The increase in partial D types accelerated dramatically after the development of the mouse hybridoma technique and the production of numerous different monoclonal anti‐Ds. According to patterns of reactivity of different partial Ds after two international workshops, the number of D epitopes reached 30. DNA techniques subsequently helped refine discrimination between partial D types. The number of described D antigen variants will surely rise after this ISBT congress and Working Party meeting. A further increase can be anticipated once NGS will provide new information and more data will come from large Asian and African populations and the polymorphic complexity of RhD and the whole Rh system will expand.