Rh Blood Group System: Key Concepts

Rh Blood Group System: Discovery, Nomenclature, and Key Concepts

Discovery and origin
- Landsteiner & Wiener discovered the Rh system in 1940 by injecting rabbits and guinea pigs with red cells from rhesus monkeys.
- Antibody produced reacted with the RBCs of about 85% of New York blood donors.
- Donors whose RBCs reacted were said to have the Rhesus factor and were called Rh positive; those whose cells did not react were Rh negative.
- Modern terminology uses Rh D positive and Rh D negative rather than Rh positive/negative; the original Rh factor was renamed D.
- The Rh system is the most complex red cell group system; it is a multiple locus system with loci that are very closely linked and multiple alleles at each locus.

Genetic Loci and Alleles

Three genetic loci on chromosome 1: C, D, E
- These loci are closely linked and encode three pairs of antigens: D/d, C/c, E/e
Genes and their encoded antigens
- D/d: D gene encodes the D antigen; d is amorphous (no detectable antigen or anti-d has ever been discovered)
- C/c: C gene encodes C antigen; c gene encodes c antigen
- E/e: E gene encodes E antigen; e gene encodes e antigen

Rh Genotype and Fisher Nomenclature

A person’s Rh genotype can be a combination of any two sets of three genes inherited as gene complexes.
Fisher nomenclature (Abbreviations)
- DCe = R1
- DcE = R2
- Dce = R0
- DCE = Rz
- dce = r
- dCe = r/
- dcE = r//
- dCE = r y
Notes on the presence/absence of D
- All shorthand forms listed above include D for Rh D positive and include d for Rh D negative where applicable.
- r and r/ and r// are the conventional symbols used to describe Rh genotypes with D absent or weakened in certain combinations.

Common Rh Genotypes and Population Frequencies

Some genotype combinations are common; others are rare, with frequencies varying by race.
Fisher nomenclature gene complexes and approximate frequencies (Caucasian vs Black American):
- DCe (R1): $ext{Caucasian} ightarrow 41\% , ext{Black American} ightarrow 8\%$
- dce (r): $ext{Caucasian} ightarrow 37\% , ext{Black American} ightarrow 25\%$
- DcE (R2): $ext{Caucasian} ightarrow 17\% , ext{Black American} ightarrow 7\%$
- Dce (R0): $ext{Caucasian} ightarrow 3\% , ext{Black American} ightarrow 49\%$
- dcE (r//): $ext{Caucasian} ightarrow 1\% , ext{Black American} ightarrow 1\%$
- dCe (r/): $ext{Caucasian} ightarrow 0.7\% , ext{Black American} ightarrow 2\%$
- DCE (Rz): $ext{Caucasian} ightarrow 0.4\% , ext{Black American} ightarrow 0.5\%$
- dCE (r y): $ext{Caucasian} ightarrow 0.001\% ext{ (very rare)}$
Common Rh genotypes in Caucasians (combinations from two parents):
- DCe/dce (R1r): $32.7\%$
- DCe/DCe (R1R1): $17.7\%$
- dce/dce (rr): $15.1\%$
- DCe/DcE (R1R2): $11.9\%$
- DcE/dce (R2r): $10.9\%$
- dCe/dce (r/R0): $2.2\%$
Rarest Rh genotypes in Caucasians:
- DCE/dCE (Rz r y): very rare
- dCE/dCE (r y r y): very rare

Nomenclatures and Naming Systems

Naming systems for Rh genotypes
- Fisher system (preferred per WHO)
- Wiener system (used in America in some contexts)
- Rosenfield system found in some literature
WHO recommendation
- WHO recommends using the Fisher system; this course uses the Fisher nomenclature.

Antigens on Red Cells and Antibody Specificity

Antigens on red cells are arranged in two sets of three antigens per cell.
- One set inherited from one parent, the other from the other parent.
Possible antigen combinations (DCE, dce, DcE, Dce, dcE, dCe, DCe, dCE) where big letters denote presence and little letters denote absence.
Example: Cde = big C, little d, little e.
Antibodies can be specific for a single antigen (e.g., anti-D, anti-C) or for combinations present on a given set (e.g., anti-DC or anti-ce).

Antibodies of the Rh System

Most Rh antibodies are IgG and are sensitizing antibodies; best detected by the Indirect Antiglobulin Test (IAT, Coombs test).
Some IgM antibodies (agglutinating) can be detected in routine testing.
Rh antibodies are immune-formed Abs produced as a result of:
- Red cell transfusion (exposure to non-self Rh antigens)
- Transplacental bleeding from infant to mother (hemolytic disease of the newborn, HDNB)
Immunogenicity order (by antigen):
- D antigen: the most immunogenic protein antigen; up to $80\%$ of Rh D negative individuals receiving Rh D positive cells will produce anti-D.
Most clinically significant Rh antibody: Anti-D.
Other antibodies (in decreasing order of frequency):
- Anti-C + D (anti-CD): reacts with cells that are C positive or D positive or both
- Anti-c
- Anti-C
- Anti-E
- Anti-f (reacts only with cells that have both c and e antigens)
- Anti-e

Clinical Significance of Rh Antibodies

All Rh antibodies can cause clinical consequences:
- Severe hemolytic transfusion reactions (HTR)
- Hemolytic disease of the newborn (HDNB)
Because Rh antibodies (especially anti-D) do not occur naturally, the purpose of Rh testing is to:
- Prevent formation of antibodies
- Detect any antibodies previously formed
Routine testing focuses on Rh D antigen; testing for other Rh antibodies is performed when blood is matched for transfusion.

Practical Aspects of Rh D Testing

All patients who may require red cell transfusions are tested for Rh D antigen only.
Rh D negative patients receive Rh D negative red cells.
Rh D positive patients may receive Rh D positive or Rh D negative red cells.

Anti-D Reagents and Testing Approaches

Types of anti-D reagents: 1) Polyclonal IgG anti-D
- Made to agglutinate Rh D positive cells by adding:
  - High molecular weight (HMW) polymer OR
  - Enzymes that digest heavy-chain disulfide bonds
- Contains multiple anti-D antibody molecules with slightly different specificities (paratopes); broad reactivity with most Rh D positive cells.
  2) Monoclonal anti-D
- Two types:
  - IgM anti-D: reacts in saline with most Rh D positive cells; used in emergency & routine screening.
  - IgM + IgG: blend of two monoclonal antibodies (agglutinating & sensitizing) used at 37°C.
Negative controls
- An immunologically inert anti-D is included with the test and contains all components except anti-D.
- With polyclonal IgG or IgM+IgG monoclonal reagents, the negative control must read 0.

Interpretation of Rh D Test Results

Example results and interpretations: 1) Patient cells + anti-D ++++ vs Patient cells + Rh D control 0
- Report as Rh D positive.
  2) Patient cells + anti-D 0 vs Patient cells + Rh D control 0
- Report as Rh D negative for recipients; further testing needed for donors.
  3) Patient cells + anti-D ++++ vs Patient cells + Rh D control ++++
- Do not report as positive (Rh D control positive invalidates result).
- Possible explanations: red cells sensitized in vivo with IgG Ab; HMW additives causing agglutination.
- Confirm by washing test cells 4x in saline and performing DAT (Direct Antiglobulin Test).
- If DAT positive, red cells are sensitized with IgG Ab. To resolve: use IgM saline agglutinating anti-D to type the tested red cells (does not contain HMW additives).
Additional practical considerations for false positives
- Agglutination may be due to a high-titer cold IgM agglutinin.
- Confirmation steps: incubate cells at 37°C to see if agglutination disappears; test patient's serum for cold agglutinin.
- If cold agglutinin suspected, wash tested red cells 4x in warm saline and repeat; this should resolve false positives.

Direct Antiglobulin Test (DAT)

Principle: DAT detects whether IgG antibodies or complement are coating RBCs in vivo (sensitization).
Procedure: Red cells from patient are washed to remove unbound Abs; antiglobulin (AHG) reagent is added.
Applications of DAT:
- Autoimmune or drug-induced hemolytic anemia
- Hemolytic disease of the newborn (HDN)
- Suspected hemolytic transfusion reaction (HTR)

Indirect Antiglobulin Test (IAT)

Principle: IAT identifies antibodies in serum.
Procedure:
- Serum (antibody-containing) is incubated with reagent RBCs to allow in vitro sensitization.
- Red cells are washed to remove unbound antibodies.
- Coombs reagent (AHG) is added to detect sensitization.
Uses of IAT:
- Antibody screening
- Antibody identification (ID) or panel testing
- Crossmatching
- Weak D (Du) identification

Weak D Antigen (Du)

Definition: Weak D is a weakened expression of the D antigen due to a low number of D antigen sites on red cells.
Causes: partial D expression or inherited fewer D receptors on red cells.
Testing implications:
- Not all anti-D reagents will agglutinate weak D cells.
- IgG anti-D will sensitize cells but may not cause visible agglutination if there is no HMW additive.
- Routine testing may classify weak D cells as Rh D negative; this is acceptable for recipients (they can receive Rh D negative blood).
- For donors, weak D cells must be classified as Rh D positive to prevent alloimmunization; Du must be detected by IAT.
Donor and recipient implications:
- Even though D antigen is weak, transfusing weak D cells into an Rh D negative recipient can stimulate anti-D production and cause a transfusion reaction.
- Therefore, all Rh D negative donors must be checked for weak D by IAT after incubation with anti-D; any positive results are designated as weak D positive and these units can only be given to Rh D positive recipients.
Detection improvements:
- Detection of weak D has improved due to better polyclonal reagents and more widespread use of monoclonal anti-D reagents, increasing identification of D-positive cells that would previously have been classified as weak D.

Indirect Antiglobulin Test (IAT) – Applications in Blood Banking

Summary of IAT uses: antibody screening, antibody identification (panel), crossmatching, and weak D identification.

Grading System for Agglutination (Role in Testing)

Grading scale for agglutination (examples shown in lab reports):
- 4+ : One solid agglutinate, no free cells (clear)
- 3+ : Several large agglutinates, few free cells (clear)
- 2+ : Many medium agglutinates, moderate number of free cells (clear)
- 1+ : Many small agglutinates, turbid appearance
- W (weak): Many tiny agglutinates not visible without a microscope; turbid, dark appearance
- 0 (negative): No agglutination (homogeneous)

References and Resources

Harmening, D.M. (2018). Modern Blood Banking & Transfusion Practices, 7th ed.
Overfield, Dowson, Hamer (2007). Transfusion Science (2nd Revised ed.) Scion Publishing
Dacie & Lewis. Practical Haematology, 9th ed. Churchill Livingstone (2001)
Norman Sellel? (examples provided in lecture) – Immunohaematology texts
Additional immunology texts listed in course references

Quick Summary of Key Concepts

Rh system is highly complex with multiple gene loci (C, D, E) and numerous antigen expressions.
The D antigen is the most immunogenic; anti-D is the most clinically significant Rh antibody.
Testing strategy focuses on Rh D for routine screening, with extended testing for other Rh antibodies during transfusion planning.
Weak D (Du) can complicate classification of donors and recipients; IAT is essential for accurate detection.
IAT and DAT are complementary tests used to identify antibodies in serum and to detect in vivo sensitization of RBCs, respectively.
Proper interpretation of test results requires consideration of potential in vivo sensitization, cold agglutinins, and the need for confirmatory DAT or alternate reagents.