Rh Blood Group System and Genetic Inheritance

• "Rh-positive" and "Rh-negative" indicate the presence or absence of the D antigen on red cells.
• Absence of the D antigen usually does not mean there are anti-D antibodies in the plasma.
• Anti-D antibody production necessitates immune red cell stimulation (transfusion/pregnancy).
• The Rh blood group system was found during investigations into transfusion reactions and hemolytic disease of the fetus and newborn (HDFN).
• In 1940, Levine and Stetson connected HDFN to an antibody in the Rh blood group system.
• The Rh system was named after experiments stimulating guinea pigs and rabbits with red cells from Rhesus macaque monkeys, which produced an antibody similar to the maternal antibody observed in HDFN cases.

Genetic Inheritance

• The current genetic theory of Rh system antigens involves two linked genes on chromosome 1: RHD and RHCE.
• The RHD gene determines D antigen expression; D-negative individuals lack genetic material at this locus.
• The RHCE gene is responsible for C, c, E, and e antigens with alleles like RHCE, RHCe, RHcE, and RHce.
• These genes code for polypeptides, explaining various Rh antigen expressions.

Biochemistry

• Genes and gene products:
  • RHD encodes the RhD protein (D antigen presence/absence).
  • RHCE encodes the RhCE protein (C/c and E/e antigen expression).
• Both genes share about 96% sequence homology and are integral transmembrane proteins in red blood cell membranes.
• RhD protein expresses the D antigen.
• RhCE protein expresses C, c, E, and e antigens through specific amino acid substitutions.
• RhD and RhCE proteins span the RBC membrane 12 times, with intracellular N- and C-terminal ends.
• Rh proteins belong to the ammonia transporter family and are associated with RBC membrane stability and gas transport.
• Antigenic epitopes are formed by conformational changes in extracellular loops, determined by specific amino acid sequences.
• Rh proteins interact with Rh-associated glycoprotein (RhAG), essential for expression in the RBC membrane.
• The Rh system consists of over 49 antigens; D, C, c, E, and e are the most clinically significant.
• C vs. c antigen: Amino acid Ser103Pro.
• E vs. e antigen: Amino acid Pro226Ala.
• Rh-negative individuals usually lack the RHD gene or have nonfunctional variants.
• Weak D, Partial D, and Del variants result from mutations or hybrid RHD-RHCE genes.

Fisher-Race vs. Wiener

• Fisher-Race Theory (CDE):
  • Inheritance: antigens inherited as a gene complex for three sets of alleles.
  • Gene Loci: D, C/c, and E/e genes inherited at separate loci.
  • Parental Contribution: Each parent contributes one haplotype (set of Rh genes). -D gene produces D antigen, etc.
  • Notation: Written as CDE (though order on chromosome is DCE).
• Wiener Theory (Rh-Hr):
  • Inheritance: alleles at one gene locus responsible for expression.
  • Gene Locus: one RH gene from each parent.
  • Alleles: eight alleles at the RH gene locus: R0, R1, R2, Rz, r, r′, r″, and ry.
  • Agglutinogen Concept: gene encodes an agglutinogen, identified by its parts or factors (Rh0, rh′, hr″).
  • Antigen Complex: agglutinogen comprises factors identifiable as separate antigens (D, C, c, E, and e).
• Translation:
  • D Antigen: R (Wiener) = D (Fisher-Race).
  • No D Antigen: r (Wiener) = no D (Fisher-Race).
  • C Antigen: 1 or ′ (Wiener) = C (Fisher-Race).
  • E Antigen: 2 or ″ (Wiener) = E (Fisher-Race).

Rosenfield and ISBT Terminologies

• Rosenfield Terminology:
  • Structure: for computerized data entry, describes phenotypic information.
  • Each antigen given a number corresponding to its assignment in the Rh system.
  • Usage: for computerized data entry, expressing a red cell's phenotype.
  • Example: Rh:1,2,−3,4,5 (D+, C+, E−, c+, e+), minus sign indicates negative result.
• International Society of Blood Transfusion (ISBT) Terminology:
  • Structure: six-digit number to each blood group specificity.
  • First three digits represent the blood group system, last three represent the antigen specificity.
  • Usage: Standardized, universally recognized system for international data exchange.
  • Example: 004002 (004 is Rh system, 002 is C antigen).

Predicting Genotype

• White Population:
  • Phenotype CcDe genotype is likely R1r (CDe/ce).
• Black Population:
  • Phenotype CcDe genotype is likely R1R0 (CDe/cDe).

Steps to Determine Genotype:

• Identify Phenotype: identify which Rh antigens are present/absent.
• Reference Common Gene Frequencies: use gene frequencies for the person's racial background.
• Infer the Genotype: based on phenotype and gene frequencies.

Weak D

• Weak D refers to red cells with weaker-than-usual D antigen expression.
• These cells may require the indirect antiglobulin test (IAT) for detection.
• Red cells that are D positive only by the IAT are referred to as weak D.

Genetic Circumstances:

• Genetic Variations: variations within the RHD gene results in altered D antigen.
  • Single Nucleotide Polymorphisms (SNPs): Changes to gene sequence affects expression.
  • Partial D Alleles: Alleles produce a weaker D antigen not fully recognized by anti-D reagents.
• Position Effect: interaction of RHCE gene with RHD gene can influence D antigen expression.
• Hybrid Genes: genes formed due to the exchange of genetic material between the RHD and RHCE genes.

Serologic Testing:

• Indirect Antiglobulin Test (IAT): may require the IAT to detect D antigen presence.
• Monoclonal Anti-D Reagents: can detect weak D antigens without IAT.
• Control Tests: A Rh or D control verifies accuracy.

Weak D Antigen Testing

• Initial testing with anti-D reagents to see agglutination directly (if no agglutination move to next stage).
• Indirect Antiglobulin Test (IAT): Incubate red cells with anti-D reagent, if D antigen is present AHG reagent will cause agglutination .
• A D control or autocontrol ensures that any positive result is because of the D antigen and the red cells are not already coated with antibodies (IgG).

Significance of Appropriate Controls:

• Validation of results: the D control ensures that the positive result from the weak D test is accurate
• Accurate donor classification: units are correctly D-positive and are transfused only to D-positive recipients.
• Avoid False positives: By using a control labs verify that a result is from a weak expression and not antibodies on the red cells.

Significance of Testing:

• Testing ensures transfusion safety, guides Rh immune globulin (RhIG) administration and prevents HDFN.
• Helps identify donors and pregnant women by ensuring they receive appropriate treatment, preventing alloimmunization, transfusion reactions, and complications (HDFN).

Cis-Product Antigens

• Result from genes encoded by the same haplotype, rather than a single gene.
• Examples: ce(f) and Ce.

G Antigen

• Present on any red cell that carries either the D or C antigen (or both).
• Only absent when a person’s red cells lack both D and C.
• Individuals who make anti-G, or those who seem to produce anti-D or anti-C, should receive red cells negative for both D and C antigens.
• Anti-G is formed in almost all cases by D-negative, G-negative patients with the genotype rr (dce).

Rhnull, Rhmod and Deletion Phenotypes

• The Rhnull is from a regulator gene (RHAG) or an amorph gene, leading to the absence of Rh antigens.
• The Rmod is similar where red cells lack most Rh antigen expression due to a modified RHAG gene.
• The D-deletion phenotype involves the deletion/inactivation of genetic material at the RHCE site.

Rh System Antibodies

• Usually IgG (particularly IgG1)-bind at 37°C
• Detection is enhanced using the indirect antiglobulin test (IAT) with high-protein, LISS, proteolytic enzymes, and PEG potentiators.
• Some Rh antibodies are IgM or occur without prior transfusion/pregnancy.
• Transfusion Reactions: antibodies cause hemolytic transfusion reactions.
• HDFN: occurs when anti-D antibodies cross the placenta and destroy fetal red cells.

LW vs. Rh

• Both have antigens and antibodies that exhibit similar serologic properties.
• Anti-LW antibodies react strongly with D-positive cells and weakly with D-negative cells.
• Genetic Location: The LW blood group system is mapped to chromosome 19, whereas the Rh blood group system is located on chromosome 1.
• Antibodies to the LW system are rare but clinically significant, Rh antibodies have more widespread clinical implications.

Anti-LW

• Anti-LW antibodies are recognized for their strong reactivity with D-positive cells and weak reactivity with D-negative cells.
• Clinical significance: they can impact blood transfusion compatibility and may require careful identification to ensure safe transfusion practices.