What is The Rh blood group?

• The Rh blood group, also known as the Rhesus blood group, is a complex blood group system found in humans. It was named after the Rhesus monkey, although this was later found to be an error. The Rh blood group has gained significant importance in the field of transfusion medicine, second only to the ABO blood group. It has particularly remained crucial in obstetrics due to its association with hemolytic disease of the newborn (HDN).
• The complexity of the Rh blood group lies in the highly polymorphic genes that encode the Rh antigens. There are two closely linked genes called RHD and RHCE, and genetic rearrangements between them have given rise to hybrid Rh genes that encode various distinct Rh antigens. Currently, 49 Rh antigens are known to exist.
• The significance of the Rh blood group stems from the high immunogenicity of the Rh antigens. In the case of the D antigen, individuals who lack this antigen will produce anti-D antibodies if they come into contact with D antigen-presenting red blood cells (RBCs). This can lead to a hemolytic transfusion reaction (HTR) in blood transfusion recipients or cause HDN when fetal RBCs carrying the D antigen are attacked by maternal anti-D antibodies. Consequently, determining Rh status is a routine practice in blood donors, transfusion recipients, and expectant mothers.
• Despite the crucial role of Rh antigens in blood transfusion and HDN, the exact physiological function of the proteins is still speculative. One proposed function is their involvement in transporting ammonium across the RBC membrane and maintaining the integrity of the RBC membrane.
• The Rh blood group system consists of proteins found on the surface of red blood cells. After the ABO blood group system, the Rh blood group is the most likely to provoke transfusion reactions. Initially, the system had 49 defined blood group antigens as of 2005, but by 2023, over 50 antigens have been identified. Among them, the five most important antigens are D, C, c, E, and e. Notably, there is no d antigen.
• The Rh(D) status of an individual is usually described with a positive (+) or negative (−) suffix following the ABO blood type. For example, someone who is A+ has the A antigen and the Rh(D) antigen, while someone who is A− possesses the A antigen but lacks the Rh(D) antigen. The terms “Rh factor,” “Rh positive,” and “Rh negative” specifically refer to the Rh(D) antigen. Antibodies against Rh antigens can lead to hemolytic transfusion reactions, and antibodies against the Rh(D) and Rh antigens pose a significant risk of hemolytic disease of the fetus and newborn.

Nomenclature of Rh blood group

• The nomenclature of the Rh blood group system has evolved over time and has two main systems associated with it: the Fisher-Race system and the Wiener system. These systems were developed based on different theories of inheritance.
• The Fisher-Race system, which is more commonly used today, employs the CDE nomenclature. According to this system, each antigen is controlled by a separate gene. For example, the D antigen is produced by the “D gene,” and so on. However, it is important to note that the hypothetical “d gene” does not actually exist.
• In contrast, the Wiener system used the Rh-Hr nomenclature. According to this theory, there is one gene at a single locus on each of the two copies of chromosome 1. Each gene contributes to the production of multiple antigens. The gene R1 was believed to give rise to the “blood factors” Rh0, rh’, and rh” (equivalent to the modern nomenclature of D, C, and E antigens), while the gene r was thought to produce hr’ and hr” (corresponding to the c and e antigens in the modern nomenclature).
• Both notations, Fisher-Race and Wiener, are used interchangeably in blood banking. However, the Fisher-Race system has become more widely used due to its simplicity. Wiener’s notation is considered more complex and cumbersome for routine use.
• With the advancement of DNA testing, it has been revealed that both theories have some degree of correctness. It has been discovered that there are two linked genes: the RHD gene, which produces a single immune specificity (anti-D), and the RHCE gene, which has multiple specificities (anti-C, anti-c, anti-E, anti-e). This finding supports Wiener’s postulate that a gene can have multiple specificities, which was initially met with skepticism. However, it has been determined that Wiener’s theory of only one gene and the Fisher-Race theory of three genes are incorrect. The CDE notation used in the Fisher-Race nomenclature is sometimes rearranged as DCE to better represent the co-location of the C and E encoding on the RhCE gene, facilitating interpretation.
• In summary, the nomenclature of the Rh blood group system has undergone developments and revisions over time. The Fisher-Race system, using the CDE notation, is commonly used today, while the Wiener system’s Rh-Hr notation is less prevalent due to its complexity. Advances in DNA testing have shown that both systems have partial correctness, with the presence of two linked genes and multiple specificities within the RHCE gene.

Antigens of the Rh blood group

The Rh blood group system consists of 49 antigens, with the most significant ones being D, C, E, c, and e. The specificity of most Rh antigens is determined by the sequence of amino acids in the proteins. These antigens are carried by proteins with unknown functions.

The RhD and RhCE proteins are transmembrane proteins that pass through the red blood cell (RBC) membrane multiple times. The RhCE protein encodes the C/c antigen in the second extracellular loop and the E/e antigen in the fourth extracellular loop. It also encodes several other Rh antigens, such as Cw and Cx. Unlike many cell surface molecules, the Rh proteins do not contain oligosaccharides (they are not glycosylated), but they are closely associated with a glycoprotein called RhAG on the RBC membrane. The Rh-RhAG complex is believed to be involved in the transport of ammonium or carbon dioxide. The RhD protein specifically encodes the D antigen.

The Rh antigens are encoded by two genes, RHD and RHCE. These genes are located adjacent to each other on chromosome 1 and share 97% identity. The D/d polymorphism most commonly arises from a deletion of the entire RHD gene. The C/c polymorphism is caused by four single nucleotide polymorphisms (SNPs) that lead to four amino acid changes, with one specific change (S103P) determining the specificity of the C or c antigen. The E/e polymorphism arises from a single SNP (676G→C) that results in a single amino acid change (A226P).

The frequency of Rh antigens varies among different populations. The D antigen is present in approximately 85% of Caucasians, 92% of Blacks, and 99% of Asians. The C antigen is found in about 68% of Caucasians, 27% of Blacks, and 93% of Asians. The E antigen is present in approximately 29% of Caucasians, 22% of Blacks, and 39% of Asians. The c antigen is found in about 80% of Caucasians, 96% of Blacks, and 47% of Asians. The e antigen is highly prevalent, being present in around 98% of Caucasians, 98% of Blacks, and 96% of Asians.

Rh phenotypes, which represent combinations of specific Rh antigens, also show variation in frequency among populations. The Rh haplotype DCe is the most common in Caucasians (42%), Native Americans (44%), and Asians (70%). The Rh haplotype Dce is most common in Blacks (44%). The Rh D-negative phenotype, lacking the D antigen, is most common in Caucasians (15%), less common in Blacks (8%), and rare in Asians (1%).

Number of antigens	49: D, C, E, c, and e are among the most significant
Antigen specificity	Protein The sequence of amino acids determines the specificity of most of the Rh antigens.
Antigen-carrying molecules	Proteins with unknown function The RhD and RhCE proteins are both transmembrane, multipass proteins that are integral to the RBC membrane. The RhCE protein encodes the C/c antigen (in the 2nd extracellular loop) and the E/e antigen (in the 4th extracellular loop), plus many other Rh antigens e.g., Cw, Cx. Unlike most cell surface molecules, the Rh proteins are not glycosylated (they do not contain oligosaccharides) but they are closely associated with a RBC membrane glycoprotein called RhAG. The function of the Rh-RhAG complex might involve transporting ammonium or carbon dioxide. The RhD protein encodes the D antigen.
Molecular basis	Two genes, RHD and RHCE, encode the Rh antigens. The Rh genes are 97% identical, and they are located next to each other on chromosome 1. The D/d polymorphism most commonly arises from a deletion of the entire RHD gene. The C/c polymorphism arises from four SNPs that cause four amino acid changes, one of which (S103P) determines the C or c antigen specificity. The E/e polymorphism arises from a single SNP (676G→C) that causes a single amino acid change (A226P).
Frequency of Rh antigens	D: 85% Caucasians, 92% Blacks, 99% Asians C: 68% Caucasians, 27% Blacks, 93% Asians E: 29% Caucasians, 22% Blacks, 39% Asians c: 80% Caucasians, 96% Blacks, 47% Asians e: 98% Caucasians, 98% Blacks, 96% Asians
Frequency of Rh phenotypes	Rh haplotype DCe: most common in Caucasians (42%), Native Americans (44%), and Asians (70%) Rh haplotype Dce: most common in Blacks (44%) Rh D-negative phenotype: most common in Caucasians (15%), less common in Blacks (8%), and rare in Asians (1%)

Antibodies produced against Rh antigens

Antibodies produced against Rh antigens are primarily of the IgG type, although some IgM antibodies can also be present. Most Rh antibodies are capable of causing hemolysis, which is the breakdown of red blood cells (RBCs). However, unlike some other antibodies, Rh antibodies rarely activate the complement system. Instead, they bind to RBCs and mark them for destruction in the spleen, leading to a process called extravascular hemolysis.

When incompatible blood transfusions occur, hemolytic transfusion reactions can occur. In the case of Rh antibodies, particularly anti-D, anti-C, anti-e, and anti-c, severe hemolytic transfusion reactions can take place. These reactions can lead to the destruction of transfused RBCs. It is important to note that these reactions are typically delayed, meaning they may not occur immediately but can manifest within a few days after the transfusion.

Rh antibodies also play a significant role in hemolytic disease of the newborn (HDN). The D antigen is responsible for approximately 50% of cases of maternal alloimmunization, where the mother’s immune system produces antibodies against the Rh antigens of the fetus. When an Rh-negative mother carries an Rh-positive fetus, she may become sensitized to the D antigen during pregnancy or delivery, leading to the production of anti-D antibodies. These antibodies can cross the placenta and attack the RBCs of an Rh-positive fetus in subsequent pregnancies, causing severe HDN.

In addition to anti-D, antibodies such as anti-c can also cause severe disease in HDN. On the other hand, antibodies like anti-C, anti-E, and anti-e generally cause milder to moderate forms of HDN. The severity of HDN depends on factors such as the specific antibody involved, its titer, and the extent of RBC destruction in the fetus.

In summary, antibodies produced against Rh antigens are predominantly of the IgG type. They can cause hemolysis, leading to the destruction of RBCs. Rh antibodies rarely activate the complement system. In transfusion reactions, severe hemolytic reactions can occur, albeit typically delayed. In the context of HDN, Rh antibodies, especially anti-D, are the most common cause, leading to significant fetal RBC destruction and subsequent complications.

Antibody type	Mainly IgG, some IgM The majority of Rh antibodies are of the IgG type.
Antibody reactivity	Capable of hemolysis Rh antibodies rarely activate complement. They bind to RBCs and mark them up for destruction in the spleen (extravascular hemolysis).
Transfusion reaction	Yes—typically delayed hemolytic transfusion reactions Anti-D, anti-C, anti-e, and anti-c can cause severe hemolytic transfusion reactions. Hemolysis is typically extravascular (1).
Hemolytic disease of the newborn	Yes—the most common cause of HDN. The D antigen accounts for 50% of maternal alloimmunization (2). Anti-D and anti-c can cause severe disease. Anti-C, anti-E, and anti-e can cause mild to moderate disease.

D-antigen

• The D-antigen, also known as the RhD antigen, is an important antigen within the Rh blood group system. Individuals can either have or not have the RhD antigen on the surface of their red blood cells (RBCs). This is indicated by the terms “RhD positive” (having the RhD antigen) or “RhD negative” (lacking the antigen), which are typically added as a suffix to the ABO blood type. In some cases, the suffix is shortened to “D pos” or “D neg,” or represented by the +/- symbol. However, the use of the +/- symbol is generally avoided in research or medical contexts due to the potential for accidental alteration or obscuring.
• The D antigen holds significant medical importance. RhD-negative individuals who receive RhD-positive RBCs through transfusion may develop alloantibodies against the D antigen. These alloantibodies can lead to severe reactions when further transfusions of RhD-positive blood are administered.
• The D antigen also poses a problem in RhD-negative mothers who carry a fetus with RhD-positive RBCs inherited from the father. During childbirth or in situations like amniocentesis, fetal RBCs can enter the maternal circulation, resulting in alloimmunization against the RhD antigen. This alloimmunization can cause hemolytic disease of the newborn in subsequent pregnancies. To prevent this, Rh (D) immunoglobulin is administered to RhD-negative mothers within 72 hours of parturition.
• Unlike the ABO blood group system, there are no natural antibodies against Rh antigens. Antibodies against Rh antigens only develop in specific situations, such as Rh-incompatible pregnancies or transfusions. Most of these antibodies are of the IgG type, with few being IgM antibodies. These antibodies are considered incomplete and can be detected in newborn blood using a direct Coombs’ test or in maternal blood using an indirect Coombs’ test. These tests help identify the presence of Rh antibodies and assist in managing potential complications related to Rh incompatibility.

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