Rh Blood Group System

Historical Overview of the Rh Blood Group System

• Discovery (End of the 1930s):

  • Levine and Stetson (1939): Documented a case involving an obstetric patient who delivered a stillborn fetus and subsequently required blood transfusions. Although her husband was the same ABO type and was selected as the donor, the patient exhibited classic symptoms of a hemolytic transfusion reaction.

  • Landsteiner and Wiener (1940): Reported an antibody produced by guinea pigs and rabbits when transfused with Rhesus monkey Red Blood Cells (RBCs). This specific antibody agglutinated $85\%$ of human RBCs.

  • Terminology Distinction: The antibody produced by humans in response to transfusion or pregnancy became known as the "Rh" group. The antibody produced by the animal models was later differentiated and named "anti-LW" (in honor of Landsteiner and Wiener).

• System Expansion:

  • By the end of the 1940s, investigations led to the discovery of five additional Rh antigens.

  • The system currently contains nearly 50 specificities, but the original five (D, C, E, c, e) remain the most clinically significant.

• Biological Function: Rh antigens are involved in the transport of ammonium across the RBC membrane.

Genetic and Biochemical Foundations

• Current Genetic Theory: Expression of all Rh antigens is controlled by two closely linked genes located on chromosome 1, which follow a pattern of codominant inheritance.

  • RHD Gene: Determines the expression of the D antigen.

  • RHCE Gene: Determines the expression of the C, c, E, and e antigens.

  • Biochemistry: Rh antigens are non-glycosylated, transmembrane protein polypeptides (integral proteins). Because they lack carbohydrates and possess complex protein structures, they are highly immunogenic.

• Alternative Genetic Theories:

  • Fisher-Race Theory: Postulates that Rh antigens are controlled by three closely linked loci: $D/d$, $C/c$, and $E/e$. In this system, "d" represents the absence of the D antigen (as there is no actual "d" antigen).

  • Wiener Theory: Postulates that Rh antigens are controlled by alleles at a single gene locus. Each of the 8 possible alleles encodes an agglutinogen (which is composed of multiple blood factors). The 8 alleles are: $R_0, R_1, R_2, R_z, r, r', r'', r_y$.

Comparison of Terminology Systems

• Equivalencies (Fisher-Race, Rosenfield, and ISBT):

  • D Antigen: Fisher-Race: D; Rosenfield: Rh1; ISBT: 004001.

  • C Antigen: Fisher-Race: C; Rosenfield: Rh2; ISBT: 004002.

  • E Antigen: Fisher-Race: E; Rosenfield: Rh3; ISBT: 004003.

  • c Antigen: Fisher-Race: c; Rosenfield: Rh4; ISBT: 004004.

  • e Antigen: Fisher-Race: e; Rosenfield: Rh5; ISBT: 004005.

  • f (ce) Antigen: Fisher-Race: ce (cis-ce); Rosenfield: Rh6; ISBT: 004006.

  • Ce Antigen: Fisher-Race: Ce (cis-Ce); Rosenfield: Rh7; ISBT: 004007.

  • $C^w$ Antigen: Rosenfield: Rh8; ISBT: 004008.

  • $C^x$ Antigen: Rosenfield: Rh9; ISBT: 004009.

  • V (ces) Antigen: Rosenfield: Rh10; ISBT: 004010.

  • G Antigen: Rosenfield: Rh12; ISBT: 004012.

• Wiener to Fisher-Race Conversion Rules:

  • $R$ indicates the presence of D; $r$ indicates the absence of D.

  • Subscripts or superscripts indicating numbers ($1$) or primes ($'$) denote C.

  • Subscripts or superscripts indicating numbers ($2$) or double primes ($''$) denote E.

  • $0$ or the absence of marks indicates $ce$.

  • $Z$ or $y$ indicates the presence of both $C$ and $E$.

  • Examples:

    • CDe/ce (Fisher-Race) corresponds to $R_1r$ (Wiener).

    • $R_1R_1$ (Wiener) corresponds to CDe/CDe (Fisher-Race).

    • Ce/ce or dCe/dce (Fisher-Race) corresponds to $r'r$ (Wiener).

    • $r'r''$ (Wiener) corresponds to dCe/dcE or Ce/cE (Fisher-Race).

• Rosenfield System: Designed for computerized data. Antigens present are listed as the number (e.g., $1$); absent antigens are preceded by a minus sign (e.g., $-3$). If a person is $D+, C+, E-, c+, e+$, the Rosenfield phenotype is $Rh:1, 2, -3, 4, 5$.

• ISBT Nomenclature: Uses a six-digit number. The first three ($004$) denote the Rh system; the last three match the Rosenfield designation (e.g., C antigen is $004002$).

Clinical Characterization of the D Antigen

• Significance: D is the most immunogenic antigen outside of the ABO system. All blood donors and recipients are routinely typed for D.

• Prevalence:

  • $85\%$ of the population is D-positive.

  • $15\%$ of the population is D-negative.

• Immunogenicity: Exposure to as little as $0.1\,ml$ of D-positive RBCs can stimulate the production of anti-D in a D-negative individual.

• Risks: Anti-D is an IgG antibody that crosses the placenta, placing babies at risk for Hemolytic Disease of the Fetus and Newborn (HDFN) in subsequent pregnancies.

Weak D Phenotypes and Testing

• Definition: RBCs that do not agglutinate directly with anti-D reagents but test positive only via the Indirect Antiglobulin Test (IAT) are classified as weak D. Note that some newer monoclonal reagents can detect weak D without IAT.

• Genetic Mechanisms for Weak D:

  1. Genetic Inheritance: Inheriting a "weaker" form of the D antigen, common in the Black/African American population (often linked to the $cDe$ haplotype). These individuals are considered Rh-positive.

  2. Position Effect (C Trans to D): The D antigen expression is weakened when the C antigen is inherited on the opposite chromosome (trans position), such as in the genotype $Ce (r')$ paired with $cDe (R_0)$. These individuals are considered Rh-positive.

  3. Partial D (Mosaic D): Individuals are missing parts (epitopes) of the D antigen. They can produce anti-D against the missing components if exposed to "whole" D-positive blood. Suspect this if a D-positive person develops anti-D that does not react with their own cells.

• Weak D Test Procedure:

  1. Label tubes for anti-D and control; add 1 drop of $2-5\%$ cell suspension.

  2. Add anti-D reagent to the test tube and Rh control to the control tube.

  3. Incubate for $15-30$ minutes at $37^{\circ}C$.

  4. Wash cells 3 times with saline.

  5. Add 1-2 drops of IgG antiglobulin reagent (AHG).

  6. Mix, centrifuge, and examine for agglutination.

  7. Check negative results with IgG-coated control cells (check cells).

• Clinical Significance of Testing:

  • Donors: AABB requires weak D testing on all initially nonreactive donor units. If positive, the unit is labeled Rh-positive.

  • Neonates: Weak D testing is performed on infants of Rh-negative mothers to determine the need for Rh Immune Globulin (RhIG).

  • Recipients: Testing is not required; patients who are initially nonreactive are classified as D-negative and receive D-negative blood.

Compound, G, and Rare Phenotypes

• Compound Antigens (cis-product): Produced when two genes are inherited on the same chromosome. Example: $f (ce)$ is present when $c$ and $e$ are in the cis position. Anti-f can cause HDFN and transfusion reactions.

• G Antigen: Present on most RBCs that carry either the D or C antigen. If a patient has anti-G, they must receive blood that is both D-negative and C-negative.

• Unusual Phenotypes:

  • D-deletion ($-D-$ or $D--$): Cells lack $Cc$ and $Ee$ antigens. They show exceptionally strong D expression. Patients can only be transfused with other D-deleted cells.

  • Rh null ($--/--$): Total absence of all Rh antigens. Caused by regulator or amorph genes. Results in membrane abnormalities, shortened RBC survival, and hemolytic anemia.

  • Rh mod: Most Rh antigens are missing or severely depleted; controlled by the $RHAG$ gene.

Summary of Less Common Antigens and Antibodies

• $C^w$: Low frequency ($2\%$ in whites); can be naturally occurring; causes HTR and HDFN.

• V (ces): Found in $30\%$ of Blacks and <1\% of whites.

• Rh29: "Total Rh" antigen present on all cells except Rh null. Anti-Rh29 is made by Rh null individuals.

• hrs: An e-like variant; associated with weak e antigen typing.

Clinical Importance of Rh Antibodies

• Characteristics: Usually RBC-stimulated (via pregnancy or transfusion). Most are $IgG1$. Agglutination is best observed at the IAT phase. Potentiators are helpful for identification.

• Dosage: Antibodies to C, c, E, and e often react more strongly with homozygous cells (e.g., cells that are $EE$ versus $Ee$).

• Complement: Rh antibodies generally do not activate complement.

• Specific Antibodies:

  • Anti-E: Can be $IgM$. Often seen in combination with weak anti-c.

  • Anti-D: Can cause severe HDFN; maternal Rh settings require Rh Immune Globulin prophylaxis.

• Transfusion Rules: If a patient has a history of an Rh antibody, they must receive antigen-negative blood even if current antibody screens are negative, due to the risk of a rapid anamnesic response.

LW Blood Group System

• Relationship to Rh: LW antigens are phenotypically similar to Rh but genetically independent.

• Reactivity: Anti-LW reacts strongly with D-positive cells and weakly with D-negative cells.

• Common Phenotype: $LW(a+b-)$.

• Clinical Significance: Antibodies are rare but clinically significant.