Rh Blood Group System

Rh Blood Group System

• A complex system where alloimmunization to Rh antigens complicates transfusions and pregnancies.
• Consists of 61 different antigenic specificities.
• Second in importance to the ABO system for transfusions.
• Rh antigens are highly immunogenic.
• Rh antibodies are produced after exposure to foreign red blood cells, unlike ABO antibodies.
• The presence of Rh antibodies can cause hemolytic disease of the fetus and newborn (HDFN) and hemolytic transfusion reactions (HTRs).
• Levine and Stetson described a hemolytic transfusion reaction in 1939.
• The reaction was in an obstetrical patient transfused with ABO-compatible blood from her husband, leading to acute hemolytic transfusion reaction (AHTR).
• An antibody from the mother’s serum reacted at 37°C and 20°C with the father’s RBCs.
• Landsteiner and Weiner described an antibody made by guinea pigs and rabbits, which were transfused with rhesus macaque monkey RBCs.
• 85% of human RBCs were agglutinated by the antibody, and it was then named anti-Rh.

Terminology Systems for Rh Factors

• Fisher-Race uses DCE terminology.
• Weiner uses RH-Hr alphanumeric terminology.
• Rosenfield and coworkers use alphanumeric terminology.
• The International Society of Blood Transfusion (ISBT) nomenclature also exists.

Fisher-Race: DCE Terminology

• Defined the five most common Rh antigens.
• Postulated antigens were produced by three closely linked genes.
• Each gene is responsible for producing one antigen on the RBC surface.
• An individual inherits a set of RH genes from each parent, one D or d, one C or c, and one E or e.
• Haplotype refers to a combination of genes inherited from one parent.
• D, C, and e genes equal a DCe haplotype.
• The pairing of maternal and paternal haplotypes determines the offspring’s genotype.
• Example genotype: Dce/Dce.
• A dash (-) represents a rare phenotype with deletions of specific genes.
• An individual with D- only has D with no C/c and E/e.
• Parentheses ( ) indicate weakened antigen expression, such as (D), (C), and (e).

Weiner: Rh-Hr Terminology

• One gene defines Rh, producing an agglutinogen with three Rh factors.
• The phenotypic expression of the haplotype is considered.
• Each factor is an antigen recognized by an antibody.
• Five common Rh antigens in the original nomenclature: Rho, rh’, rh”, hr’, and hr”.
• In modified nomenclature, an agglutinogen is described by a letter and symbol based on factor presence.
• R denotes the presence of D antigen.
• r indicates the absence of D antigen.

Rosenfield and Coworkers: Alphanumeric Terminology

• A number is assigned to each antigen of the Rh system by order of discovery or recognized relationship.
• Has no genetic basis and was not based on a theory of Rh inheritance.
• Demonstrates the presence or absence of the antigen on the RBC.
• A minus sign (-) designates the absence of the antigen.
• If an antigen is not phenotyped, no number will appear in the sequence.
• DCEce is numbered 1,2,3,4,5.
• Rh: 1,2,3,-4,-5 is D+, C+, E+, c-, and e-.
• If a sample is not tested for e, the Rh is 1,2,3,-4.

The International Society of Blood Transfusion Committee

• Establishes a uniform nomenclature which is eye and machine readable while keeping with genetic basis.
• Adopted a six-digit number for each authenticated antigen belonging to a blood group system.

Genetics of Rh

• RHD and RHCE are two closely linked genes on chromosome 1 which control Rh protein expression.
• RHD codes for the presence or absence of the RhD protein.
• RHCE codes for RhCe, RhcE, Rhce, or RHCE proteins.
• They are inherited in a codominant manner.
• The RHAG gene is located on chromosome 6.
• Encodes for a polypeptide similar in structure to the Rh proteins, but is glycosylated.
• RhAG is termed a coexpressor for Rh antigens to be present however doesn't express any Rh antigens by itself.

Rh-Positive and -Negative Phenotypes

• RH genes are inherited as codominant alleles.
• Two RHCE genes are inherited, in addition to the RHD gene.
• Rh-negative phenotypes mean RBCs lack detectable D antigen.
• The most common Rh-negative phenotype results from the complete deletion of the RHD gene.
• Individuals possess no RHD gene but inherit two RHCE genes.

Most Probable Genotypes

• Serologic reactions determine most probable genotypes (MPG) for Caucasian and African American populations.
• Prevalence of RH haplotypes is important to predict correct MPG.
• If Caucasian, the genotype is R0r.
• R0 haplotype is 4% prevalent.
• r haplotype is 37% prevalent.
• If African American, most likely genotype is R0R0.
• R0- haplotype is 44%.
• Differences must be remembered when locating compatible blood for recipients with unusual or multiple Rh antibodies.

Rh Antigens

• Antigens are non-glycosylated protein on RBC membrane.
• Reside on transmembrane proteins and are an integral part of the RBC membrane.
• Gene products of RHD and RHCE are similar, encoding proteins of 416 amino acids that transverse the cell membrane 12 times.
• Proteins differ by 32 to 35 amino acids.
• Amino acid position 103 determines C or c expression.
• Position 226 differentiates E from e.
• Hughes-Jones and coworkers measured the number of D antigen sites on various Rh phenotypes.
• A D- (deletion) has the greatest number of D antigen sites.
• R2R2 is a commonly encountered Rh genotype.
• Found only on RBCs and are not soluble or expressed on other cells.
• Well-developed at birth, can cause HDFN.
• Immunogenic exposure to foreign antigens via transfusion/pregnancy can cause immune response, producing corresponding antibodies.
• D, C, E, c, and e are five common Rh antigens.
• D antigen does not have an allele.
• C and c; E and e are allelic.

D Antigen

• Most potent antigen
• Rh-negative individuals lack the entire RhD protein.
• Exposure to less than 0.1 mL of Rh-positive blood can stimulate antibody production in Rh-negative individuals.
• "d" denotes the absence of the D antigen.
• Only the presence of D is routinely tested.

Weak D Antigen

• Individuals have RBCs with variations in the quantity/specificity of D antigen epitopes, resulting in weakened D antigen expression.
• Noted when initial anti-D testing is negative or ≤ 2+, but detectable via indirect antiglobulin testing (IAT).
• Individuals with RBCs carrying weaker D antigen can produce anti-D if missing epitopes of the D antigen.
• Genetic weak D is the weak expression of D antigen.
• D antigens are complete but decreased in quantity.
• Partial D means that “D” antigen is a mosaic structure composing of 4 fragments and is also known as D mosaic
• Weakened D expression is a result of one or more D epitopes within entire D protein missing/altered.
• Alloantibody can be made to missing fraction/fragment.
• Partial D individual must receive Rh negative blood.

C in trans to RHD

• "C" is in trans position to "D" which is opposite haplotype.
• E.g. Dce/dCe
• Also known as position effect.
• The allele carrying RHD is in trans to C.
• Genes in transposition can weaken the trait's expression.

Rh Antibodies

• Immune antibodies are produced in humans via antigen exposure through transfusion/pregnancy.
• IgG reacts at 37°C, implicating HDFN/HTR.
• IgG1 and IgG3 subclasses are of greatest clinical significance.
• The RES rapidly clears RBCs coated with IgG1 and IgG3 from circulation.
• Complement doesn’t bind without two IgG attached to an RBC antigen in close proximity.
• Hemolysis is primarily extravascular.
• May show dosage-reacting preferentially with RBCs, therefore possessing double-dose Rh antigens.
• Rh antibodies are formed by pregnant women, they cross the placenta and can coat fetal RBCs carrying a corresponding antigen.
• IgG can traverse placenta.
• Rh antigens are well developed early in fetal life.
• Positive Direct antiglobulin test (DAT).

Rh Typing Reagents

• Saline (IgM) reagents are low-protein based.
• Limited availability and have a high cost of production with lengthy incubation time.
• High-protein (IgG anti-D) optimized reactivity with potentiators added.
• Potentiators reduce incubation time and give ability to perform weak D testing and slide typing.
• Presence of potentiators and a high protein concentration increases the likelihood of false-positive reactions.
• Chemically modified reagents generate fewer false-positive tests.
• Chemically modified reagents suspend in a lower-protein medium and replaced the saline anti-D reagents.
• When samples test AB Rh-positive or when the Rh test is performed by itself, a separate saline control or 6% to 8% albumin control must be used to ensure true agglutination and not spontaneous.
• Monoclonal reagents are not human-derived which lacks the potential for transmitting infectious diseases.
• Monoclonal reagents have narrow specificity and require a combinations of monoclonal anti-D reagents from different clones to ensure reactivity.

Rh typing

• D antigen is detected on RBCs.
• WB or RBCs in suspension can be used to test the specimen.
• Forward typing alone is employed, as antibodies are not naturally occurring.

Rh Deficiency Syndrome

• Rhnull Individuals lack all Rh antigens on their RBCs.
• Designated as (---/---) in Fisher-Race.
• Designated as RH:-1,-2,-3,-4,-5 in Rosenfield nomenclature
• Results in mild compensated hemolytic anemia, reticulocytosis, stomatocytosis, a slight-to-moderate decrease in hemoglobin and hematocrit levels, increase hemoglobin F, a decrease in serum haptoglobin, and may have elevated bilirubin
• If exposed to normal Rh cells through transfusion or pregnancy, can produce a potent antibody, anti-Rh29, which reacts with all cells except for those that are Rhnull.
• Rhmod have a partial suppression of RH gene expression caused by mutations in the RHAG gene.
• Exhibit RBC abnormalities similar to those with the Rhnull syndrome; however,clinical symptoms are usually less severe and rarely clinically remarkable

Unusual Phenotypes and Rare Alleles

• CW results from a single amino acid change most often found on the RhCe protein.
• f (ce) is when both c and e are present on the same haplotype and is called a compound antigen.
• rhi(Ce) is considered a compound antigen present when C and e are on the RhCe protein.
• G is an antigen present on most D-positive and all C-positive RBCs.
  • Rh17 (Hr0) is an antigen present on all RBCs with common Rh phenotypes (e.g., R1R1, R2R2, rr).
• The ff: are all low-prevalence antigens associated with a specific category of partial D. These low prevalence antigens result from the formation of the hybrid proteins seen in individuals with partial-D phenotypes.
  • Rh23 (AKA Wiel and Dw) is an antigenic marker for category Va partial-D.
  • Rh30 (AKA God or Door) is a marker for partial DIVa.
  • Rh40 (AKA Tar or Targett) is a marker for partial DVII.
  • Rh52 Rh52 or BARC is associated with some partial-DVI types.
• Most often found in whites and is associated with rare variant haplotype R0Har is Rh33(Har). R0Har gene codes for normal amounts of c, reduced amounts of e, reduced f, reduced Hr0, and reduced amounts of D antigen written as (D)c(e)
• A low-prevalence antigen associated with a variant of the R1[D(C)(e)] haplotype is Rh32 and is called R=N
• A low-prevalence antigen on a variant Rhce protein. Very low prevalence found in African descent individuals is the Rh43 (crawford) phenotype.
• Individuals who possess two altered RHCE genes may have a phenotype of e- positive but produce antibodies behaving as anti-e are the e variants.
• A Low prevalence in the Caucasian population but are more prevalent among African Americans are the V and VS phenotypes. These antigens can be used as predictors of an individual's ethnic background because of this difference inprevalence.
• This phenotype results from individuals possessing normal RHD gene(s) and hybrid RHCE-RHD-RHCE in which the Rhce protein is replaced with RhD: Deletions.