Chapter 07 The Rh Blood Group System

• The chapter describes in detail the Rh blood group system.
• It is imperative to have a basic understanding of Rh, as RhD typing is a critical component of pretransfusion testing.
• Clinically important Rh antibodies are relatively common in pregnancy and in patients requiring blood transfusion.
• The term Rh refers to a specific red blood cell (RBC) antigen (D) and to a complex blood group system currently composed of over 50 different antigenic specificities.
• Scientists have spent years unraveling the complexities of the Rh blood group system from its serology to its mode of inheritance, genetic control, and the biochemical structure of the Rh antigens.
• Rh-specific antigens reside on proteins, versus the carbohydrate antigens ABO and Hh.
• Rh is the second most important blood group system in transfusion, as the Rh system antigens are very immunogenic.
• Unlike ABO antibodies that are typically found in individuals who lack the corresponding antigen, Rh antibodies are produced only after exposure to foreign red blood cells.
• Once present, they can produce significant hemolytic disease of the fetus and newborn (HDFN) as well as hemolytic transfusion reactions.
• The terms Rh-positive or positive and Rh-negative or negative are routinely used by the public and by experts in the field when referring to blood type.
• Rh-positive indicates that an individual's red blood cells possess one particular Rh antigen, the D antigen, on their red blood cells.
• Rh-negative indicates that the red blood cells lack the D antigen.

History

• Before 1939, the only significant blood group antigens recognized were those of the ABO system.
• Transfusion medicine was thus based on matching ABO groups.
• Despite ABO matching, blood transfusions continued to result in morbidity and mortality.
• As the 1930s ended, two significant discoveries were made that would further the safety of blood transfusion and eventually define the most extensive blood group system known.
• It began when Levine and Stetson described a hemolytic transfusion reaction in an obstetrical patient.
• After delivering a stillborn infant, this woman required transfusions.
• Her husband, who had the same ABO type, was selected as her donor.
• After transfusion, the recipient demonstrated classic symptoms of an acute hemolytic transfusion reaction (AHTR).
• Subsequently, an antibody was isolated from the mother's serum that reacted both at 37°C and at 20°C with the father's RBCs.
• It was postulated that the fetus and the father possessed a common factor the mother lacked.
• While the mother carried the fetus, she was exposed to this factor and subsequently produced an antibody that showed positive reactivity when tested against the transfused RBCs from the father.
• One year later, Landsteiner and Wiener reported on an antibody made by guinea pigs and rabbits when they were transfused with Rhesus macaque monkey RBCs.
• This antibody, which agglutinated 85% of human RBCs, was named Rh after the Rhesus monkey.
• Another investigation by Levine and coworkers demonstrated that the agglutinin (antibody causing direct agglutination of RBCs) causing the hemolytic transfusion reaction and the antibody described by Landsteiner and Wiener appeared to define the same blood group.
• Many years later, the two antibodies mentioned above were recognized as different.
• The name Rh was retained for the human-produced antibody, and anti-Rhesus formed by the animals was renamed anti-LW in honor of those first reporting it (Landsteiner and Wiener).
• Further research resulted in defining Rh as a primary cause of hemolytic disease of the fetus and newborn (HDFN, also called erythroblastosis fetalis) and a significant cause of hemolytic transfusion reactions.
• Continued investigation showed additional blood group factors were associated with the original agglutinin.
• By the mid-1940s, five antigens made up the Rh system.
• Today the Rh blood group system contains over 57 different specificities and continues to grow in number as new genetic mutations are discovered.

Terminology

• Terminologies used to describe the Rh system are derived from four sets of investigators.
• Two terminologies are based on postulated genetic theories of Rh inheritance.
• The third common terminology used describes only the presence or absence of a given antigen.
• The fourth is the result of the combined efforts of the International Society of Blood Transfusion (ISBT) Committee on Terminology for Red Cell Surface Antigens.
• The molecular basis of the Rh blood group system as we know it today is described in detail in the "Molecular Genetics" section after the discussion of nomenclature to provide context to the terminology used.

Fisher-Race: DCE Terminology

• In the 1940s, Fisher and Race were investigating antigens found on human RBCs, including the newly defined Rh antigen.
• They postulated that the antigens of the system were produced by three closely linked sets of alleles.
• Each gene was responsible for producing a product (or antigen) on the RBC surface.
• Each antigen and corresponding gene were given the same letter designation (when referring to the gene, the letter is italicized).
• Fisher and Race named the antigens of the system D, d, C, c, and E, e. Now it is known that “d” represents the absence of D antigen; however, the term continues to be utilized with Fisher-Race terminology as a placeholder.
• The phenotype (antigens expressed on the RBC detected by typing) of a given RBC is defined by the presence of D, C, c, E, and e expression.
• The gene frequency in the Caucasian population for each Rh antigen is given in Table 7-1, and the Rh haplotype (the complement of genes inherited from either parent) frequencies are given in Table 7-2. Notice how the frequencies vary with ethnic background.
• According to the Fisher-Race theory, each person inherits a set of Rh genes from each parent (i.e., one D or d, one C or c, and one E or e)
• Because Rh genes were thought to be codominant, each inherited gene expresses its corresponding antigen on the RBC.
• The combination of maternal and paternal haplotypes determines one's genotype (the Rh genes inherited from each parent) and dictates one's phenotype.
• An individual's Rh phenotype is reported as DCE rather than CDE because Fisher postulated that the C/c locus lies between the D/d and E/e loci.
• This information is based on frequencies of the various gene combinations.
• It is essential to remember that d does not represent an antigen but simply represents the absence of D antigen. C, c, E, and e represent actual antigens recognized by specific antibodies.
• There has never been an antibody that recognizes d antigen, supporting the fact that d antigen does not exist.
• For many students and working laboratory scientists, the Fisher-Race terminology represents the easiest way to think about the five major Rh system antigens, but it has shortcomings.
• Many antigens assigned to the Rh blood group system were given names using a variety of terminologies.
• As the number of Rh antigens continues to grow, the original Fisher-Race terminology is becoming too limiting.
• In very rare instances, an individual may fail to express any allelic antigen at one or both Rh loci; that is, a person may lack E and e, or all CcEe antigens.
• The probable genotype for the Rh (D)-positive person exhibiting a deletion phenotype such as these is written DC- or Dc-, or D−. A deletion of Cc with Ee has not been reported.
• The person expressing no Rh antigens on the RBC is said to be Rhnull, and the phenotype may be written as —/—. Weakened expression of all Rh antigens of an individual has also been reported.
• These individuals are said to have the Rhmod phenotype. Placing parenthesis around (D), (C), and (e) indicates weakened antigen expression.

Wiener: Rh-Hr Terminology

• In his early work defining the Rh antigens, Wiener believed there was one gene responsible for defining Rh that produced an agglutinogen containing a series of blood factors.
• According to Wiener, this Rh gene produced at least three factors within an agglutinogen.
• The agglutinogen may be considered the phenotypic expression of the haplotype.
• Each factor is an antigen recognized by an antibody. Antibodies can recognize single or multiple factors (antigens).
• Wiener's terminology is complex and unwieldy; nevertheless, many blood bankers use modified Wiener terminology interchangeably with other nomenclatures.
• This terminology allows one to convey Rh antigens inherited on one chromosome or haplotype and makes it easier to discuss a genotype.
• A medical laboratory scientist conveying a probable genotype to a coworker would have to say DcE/DcE. However, R2R2 is much easier to verbally communicate.
• Fisher-Race nomenclature may be converted to Wiener nomenclature and vice versa.
• It is important to remember that an agglutinogen in Wiener nomenclature actually represents the presence of a single haplotype expressing three different antigens.
• When describing an agglutinogen, the uppercase R denotes the presence of the original factor, the D antigen.
• The lowercase r indicates the absence of D antigen.
• The presence of uppercase C is indicated by a 1 or a single prime ('). Lowercase c is implied when there is no 1 or' indicated.
• The presence of E is indicated by the Arabic number 2 or double prime ("). Lowercase e is implied when there is no 2 or " indicated.
• When both C and E are uppercase, the letter z or y is used.
• Italics or superscripts are used when describing Rh genes in the Wiener nomenclature (i.e., R¹ or R2, R¹ or R2).
• Standard type is used to describe the gene product or agglutinogen.
• Subscripts are used with the uppercase R and superscripts with the lowercase r (i.e., R₁ or R2 or r').
• Phenotypes of Rhnull and Rhmod are written as stated.
• The genotype for the Rhnull that arises from an amorphic gene at both Rh loci is written as mand pronounced "little r double bar."
• When referring to the Rh antigens (or blood factors) in Wiener nomenclature, the single prime (') refers to either C or c and the double prime (") to either E or e.
• If the r precedes the h (i.e., rh' or rh"), this refers to the C or E antigens, respectively.
• When the h precedes the r, this refers to either the c (hr') or e (hr") antigen. Rho is equivalent to D.
• There is no designation for the absence of D antigen exist in in the Wiener nomenclature.
• By using these designations, the laboratorian should be able to recognize immediately which antigens are present on the RBCs described.
• However, it is difficult to use the Wiener nomenclature to adequately describe additional alleles within an agglutinogen.
• Because of this, many of the more recently described antigens of the Rh system have not been given Rh-Hr designations.

Rosenfield and Coworkers: Alphanumeric Terminology

• As the Rh blood group system expanded, it became more difficult to assign names to new antigens using existing terminologies.
• In the early 1960s, Rosenfield and associates proposed a system that assigns a number to each antigen of the Rh system in order of its discovery or recognized relationship to the Rh system.
• This system has no genetic basis, nor was it proposed based on a theory of Rh inheritance, but it simply demonstrates the presence or absence of the antigen on the RBC.
• A minus sign preceding a number designates the absence of the antigen.
• If an antigen has not been typed, its number will not appear in the sequence.
• An advantage of this nomenclature is that the RBC phenotype is thus succinctly described.
• For the five major antigens, D is assigned Rh1, C is Rh2, E is Rh3, c is Rh4, and e is Rh5.
• The numeric system is well suited to electronic data processing. Its use expedites data entry and retrieval.
• Its primary limiting factor is that there is a similar nomenclature for numerous other blood groups such as Kell, Duffy, Kidd, Lutheran, Scianna, and more.
• When using the Rosenfield nomenclature on the computer, one must use both the alpha (Rh:, K:) and the numeric (1, 2, -3, etc.) to denote a phenotype.

International Society of Blood Transfusion Committee: Updated Numeric Terminology

• As the world of blood transfusion began to cooperate and share data, it became apparent there was a need for a universal language.
• The International Society of Blood Transfusion (ISBT) formed the Committee on Terminology for Red Cell Surface Antigens.
• Its mandate was to establish a uniform nomenclature that is both eye- and machine-readable and is in keeping with the genetic basis of blood groups.
• The ISBT adopted a six-digit number for each authenticated antigen belonging to a blood group system.
• The first three numbers represent the system and the remaining three the antigenic specificity.
• Number 004 was assigned to the Rh blood group system, and then each antigen assigned to the Rh system was given a unique number to complete the six-digit computer number.
• When referring to individual antigens, an alphanumeric designation similar to the Rosenfield nomenclature may be used.
• The alphabetic names formerly used were left unchanged but were converted to all uppercase letters (e.g., Rh, Kell became RH, KELL).
• The phenotype designation includes the alphabetical symbol that denotes the blood group, followed by a colon and then the specificity numbers of the antigens defined.
• A minus sign preceding the number indicates that the antigen was tested for but was not present.
• Tables summarize the data presented, and include probable genotypes based on the antigens found in selected RBC populations.
• Tables also correlates Rh phenotypes with the most probable or predicted genotype in a designated population.
• Results of typing do not define genotype, only phenotype.
• Determining probable or predicted genotypes was useful for parentage studies, also known as relationship testing, and for population studies. Other molecular methods are proving more powerful today.
• Probable genotypes are useful in predicting the potential for HDFN in offspring of Rh-negative women with anti-D; however, molecular testing can be performed to confirm whether the father possesses one or two copies of the RHD gene.
• There are substantial differences in phenotypes and predicted genotypes of various populations
• These differences must be remembered when trying to locate compatible blood for recipients with unusual or multiple Rh antibodies.

Molecular Genetics

• Several theories have been described to explain genetically the results of serologic and biochemical studies in the Rh system.
• Two theories of Rh genetic control were initially postulated:
  • A single gene produces a single product that contains separately recognizable factors
  • The Rh locus contains three distinct genes that control production of their respective antigens
• Two RH genes, RHD and RHCE, control expression of Rh antigens:
  • The gene RHD codes for the presence or absence of the RhD protein
  • The second gene RHCE codes for either RhCe, RhcE, Rhce, or RhCE proteins
• RHD and RHCE genes each have 10 exons and are 97% identical.
• Another gene important to Rh antigen expression is RHAG, and it resides on chromosome 6.
• The product of this gene is Rh-associated glycoprotein (RhAG), and the polypeptide is glycosylated (carbohydrates attached).
• Within the RBC membrane, it forms complexes with the Rh proteins and is termed a coexpressor:
  • It must be present for successful expression of the Rh antigens
  • By itself, this glycoprotein does not express any Rh antigens
• When mutations in the RHAG gene occur, it can result in missing or significantly altered RhD and RhCE proteins, affecting antigen expression.
• In rare instances, individuals express no Rh antigens on their RBCs:
  • They are said to have the Rhnull phenotype
  • RHAG has been assigned as a blood group system

Rh-Positive and Rh-Negative Phenotypes

• Rh Genes are inherited from the parents like codominant alleles.
• Rh Positive Phenotypes result in expression of RhD antigen, the individual is typed Rh-positive. In addition to the RHD gene(s), two RHCE genes are inherited, one from each parent
• Numerous mutations in the RHD gene cause weakened expression of the RhD antigen detected in routine testing.
• Rh negative individuals can arise from at least three different mutations:
  • European ethnicity: deletion of the RHD gene, they possess no RHD gene but have inherited two RHCE genes
  • African ethnicity: unusual form of the RHD gene called RHD pseudogene or RHDy, does not produce RhD protein and has sequence identical to RHD gene
  • Asian Ethnicity: a mutation thatalters the RHD gene, an individual types as D-negative.
  • Termed Del
• Numerous mutations have been described in the RH genes:
  • Greater than 150 alleles have been determined in the RHD gene, and greater than 60 alleles have been found in the RHCE allele and the number continues to grow
• The product of RH genes are nonglycosylated proteins:
  • This means no carbohydrates are attached to the protein
• Rh antigens reside on transmembrane proteins and are an integral part of the RBC membrane.
• The gene products of RHD and RHCE are remarkably similar in that both encode for proteins composed of 416 amino acids that traverse the cell membrane 12 times.
• . Their proteins differ by only 32 to 35 amino acids:
  • Amino acid position 103 is important in determining C or c expression and position 226 differentiates E from e
• According to investigations, In comparison with ABO and Kell (K) blood groups, Aı cells possess approximately 1 × 106 A antigens, whereas RBCs possessing a double-dose expression of the K antigen have 6,000 K sites
• Hughes-Jones and coworkers measured the number of D antigen sites on a variety of Rh phenotypes showing the greatest number antigen sites on cells of the rare Rh phenotype D
• RhD and RhCE proteins and RhAG are exclusively on red blood cells:
  • As they are transmembranes, plays a role in maintaining the structural integrity of red cells
  • Based on their structure, it appears they may also be transporters. Westhoff and colleagues showed they may have a role in transporting ammonia.
  • An alternative hypothesis is that they may be CO2 transporters

Weak D: Variations of D Antigen Expression

• Rh-positive RBC samples are typed for the D antigen and are expected to show strong positive reactivity with anti-D reagents.
• Some individuals have RBCs that possess D antigen that requires an indirect antiglobulin test to detect the presence of D antigen.
• RBCs carrying weaker D antigen were referred to as having the Du when the investigators thought it was a new antigen.
• It was later proven that this was not a new antigen since individuals did not procedure anti-Du; they produce anti-D.
• Now these individuals with altered D antigen are categorized into different phenotypes defined as:
  • Weakened D due to C in trans to RHD
  • Weak D
  • Partial D
  • Del
  • D epitopes on their RhCE protein
• 1%-2% of people with European ancestry possess altered form of D antigen while altered D antigen occurs more often in individuals of African descent but exact prevalence is not known.
• Three ways that weaken the D antigen are:
  • C in Trans to RHD
  • Weak D
  • Partial D
• Weak d antigen is when the allele carrying RHD is trans to the allele carrying C: normal Rh antigen on RBC but steric arrangement appears to interfere with the expression of D antigen.
• Weak D individuals who were typed D- exhibit molecular mutations in RHD gene causing changes to amino acids present in transmembrane or intracellular region of RhD protein.
• Partial d antigens have one or more D epitopes in the D protein either missing or altered:
  • Some red cells with partial D antigen may type weaker than expected, others may show normal typing
  • The theory states, individuals lack one (or more) pieces, or epitopes, or the total D antigen, alloantibody can be made to the missing epitope(s) if exposed to RBCs that possess the complete D antigen
• The result for partial-D antigens is hybrid genes from Portions of the RHD gene being replaced by portions of the RHCE gene, resulting protein contains only RhD and RhCE and only produce antibody to RhD
• The phenotype exhibiting extremely low number of antigen sites often undetectable by anti-D agents is classified as Del

Detection of Rh Antibodies and Antigens

• Basic concepts:
  • Rh antibodies are fairly straightforward to detect and identify as compared to understanding the molecular genetics and biochemistry of the Rh blood group system
  • Further discussion on detecting antibodies made by individuals to Rh antigens follows.
• Rh Antibodies:
  • Rh antibodies are generally non-saline IgM and react optimally and are best tested post antiglobulin testing with IgG at 27degrees C.
  • Rh antibodies are immunogenic and produce results in 120 days after a primary exposure of 2-7 days after secondary
  • Rh antibodies IgG1 and IgG3 are clinically significant due to the reticuloendothelial system and should be considered
  • Because Rh antibodies are IgG they may traverse the placenta causing HDFN and therefore are important
• Rh Typing Reagents:
  • reagents type or anti-D may be derived of various types, or low based, or chemically modified to allow quick determination of accurate ABO results.
  • Saline reactive reagents used to detect IgG that may cause false positives like warm antibodies
  • High-Protein anti-D reagents developed in the 1940s contained human plasma in order to test weak-D typing, reduced incubation time
  • Chemically modified reagents use broken disulfide bonds to allow more accurate reading
  • Monoclonal antibodies are used to detect antigens
• Clinical Considerations:
  • Determining an individual RhD type and testing to identify antibodies has been reviewed, is done in the context of clinical implications and incompatibilities.
• Transfusion Reactions:
  • Rh antigens are highly immunogenic and should be carefully considered for use to prevent an adverse transfusion reaction, exposure results in antibody productions from 2-120 days after exposure and can illicit fever, hyperbilirubinemia, less haptoglobin.
• False positives
• Falso negatives result from using reagent samples with the incorrect protocol or expired samples.
• Clinical notes state it is important to carefully distinguish and evaluate obstetric patients for anti-G antigen, a candidate blood for transufions can come form -D, -c-G.

The Rh deficiency syndrome, types of Rhnull and Rhmod

• People who fail to express RH antigens in the RBC due to a mutation in the regulatory, RHAG gene.
• A deficiency of the Rh antigens in the red cells cause Rhnull syndrome the following symptoms:
• Mild hemolytic anemia
• Reticulocytosis
• Presence of stomatocytes on the blood smear

Unusual Phenotypes and Rare Alleles:

• This section describes the variants Cw, f(ce), G, some Rh:13, Rh:14, Rh:15, and Rh:16, Rh17, Rh:23, Rh:30, and Rh:40, Rh har, as well as the Landsteiner-Weiner Blood Group System.
• All of these different notations are described in each section, with specifics that affect serology
• Cw was thought to be an allele and is now proven to be an antithetical presence of a high prevalence MAR.
• Is only seen in about 2% of whites and is very rare.
• The f antigen (ce) exists when the antigen C and E are both present on the same haplotype (called a “cis-product”
• However it’s also simply the antigen from conformational changes in the Rhce protein.
• G antigen exists on most cells, the antibody functions similar to anti-D testing.
• RH:13, Rh:14, Rh:15 and Rh 16 Define the four different parts of the D “mosaics” and are now obselete
• RH 17 results in anti-Hro anti body.
• RH23, Ro, Rh 40; result from the hybrid form of partial -D phenotypes.
• Rh Har is rare allele on the D antigen
• Most of the antigens result from specific protein confirmations resulting in specific serologic findings (but no specific action necessary)
• Landesteiner-Weiner blood group is still detected by some antibodies. However is distinct.