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Rh

The Rh blood group system (ISBT 004) stands second only to ABO in clinical importance, largely due to the high immunogenicity of the D antigen and its role in severe Hemolytic Disease of the Fetus and Newborn (HDFN) and transfusion reactions. Unlike carbohydrate-based systems, Rh antigens (D, C, c, E, e being the most significant) are integral parts of complex transmembrane proteins encoded by the closely linked RHD and RHCE genes on chromosome 1. Understanding the biochemistry of these proteins and their variants is crucial for safe transfusion practice

Genetics Recap: The Foundation

Remember those two highly similar (homologous) and closely linked genes on chromosome 1?

  • RHD Gene: Codes for the RhD protein. Determines RhD positive or negative status
  • RHCE Gene: Codes for the RhCE protein. Polymorphisms (variations) in this gene determine the expression of C vs c and E vs e antigens
  • Linkage: Because they are so close, we inherit them together as a haplotype (e.g., DCe, dce, DcE). We get one haplotype from each parent
  • RHAG Gene (Rh-Associated Glycoprotein): Located on chromosome 6. This gene is crucial! It codes for a “coexpressor” or “support” protein (RhAG) that is essential for trafficking and integrating the RhD and RhCE proteins into the red blood cell membrane. No functional RhAG = no RhD/RhCE expression (one cause of the Rhnull phenotype)

Biochemistry: The Rh Proteins

Unlike ABO and Lewis antigens which are carbohydrates built by enzymes, Rh antigens are integral parts of proteins that span the red blood cell membrane

  • RhD and RhCE Proteins
    • Highly similar in structure (about 92% identical amino acid sequence)
    • Large, transmembrane proteins that loop through the RBC membrane 12 times
    • They are not glycosylated (no sugar chains attached), which is unusual for many red cell surface proteins
    • Function isn’t fully confirmed, but they are thought to be involved in membrane integrity and potentially act as transporters (perhaps for ammonia or CO2), though this is still debated
    • They exist in the membrane as part of a protein complex, primarily associated with RhAG. Other proteins like LW, Glycophorin B, CD47, and Band 3 might also be part of this complex, which likely helps maintain RBC shape and structure
  • Rh-Associated Glycoprotein (RhAG)
    • Also a transmembrane protein with 12 loops, structurally similar to RhD/RhCE but coded by a different gene (RHAG) on a different chromosome
    • It IS glycosylated
    • Essential for the expression of RhD and RhCE proteins on the cell surface. It acts like a scaffold or escort service to get them properly inserted into the membrane

The Major Rh Antigens: D, C, c, E, e

These five antigens are the most important serologically. Their presence or absence is determined by the specific RhD and RhCE proteins expressed

  • D Antigen
    • The most immunogenic Rh antigen
    • Presence or absence determines Rh positive (D+) or Rh negative (D-) status
    • Located on the RhD protein. If the RHD gene is present and functional, the RhD protein is made, and the person is D+
    • If the RHD gene is absent (like the common deletion in Caucasians) or non-functional (due to mutations), no RhD protein is made, and the person is D-
    • There is no “d” antigen.: The lowercase “d” simply indicates the absence of the D antigen
  • C/c Antigens
    • Antithetical pair (coded by different alleles at the same genetic locus within RHCE)
    • Located on the RhCE protein
    • Differ by a single amino acid change, determined by SNPs in the RHCE gene. Specifically, Serine at position 103 corresponds to C, while Proline at position 103 corresponds to c
    • An individual can be homozygous (CC or cc) or heterozygous (Cc)
  • E/e Antigens
    • Another antithetical pair
    • Also located on the RhCE protein
    • Differ by a single amino acid change at a different location on the protein, determined by SNPs in the RHCE gene. Specifically, Proline at position 226 corresponds to E, while Alanine at position 226 corresponds to e
    • An individual can be homozygous (EE or ee) or heterozygous (Ee)
  • Combined Expression: The RhCE protein expresses either C or c and either E or e on the same molecule, depending on the inherited RHCE allele (haplotype). For example:
    • The DCe haplotype codes for RhD protein and an RhCE protein expressing C and e
    • The DcE haplotype codes for RhD protein and an RhCE protein expressing c and E
    • The dce haplotype codes for no RhD protein and an RhCE protein expressing c and e

Nomenclature Snippet

  • Fisher-Race (DCE): Describes the antigens (D, C/c, E/e). Genotypes written based on haplotypes (e.g., DCe/dce)
  • Wiener (Rh-hr): Postulated a single gene with multiple alleles. Uses shorthand (e.g., R1 = DCe, r = dce, R2 = DcE, R0 = Dce). Less intuitive biochemically but still widely used in notation
  • ISBT Numerical: Standardized system (Rh is 004). D=RH1, C=RH2, E=RH3, c=RH4, e=RH5

Variations in D Antigen Expression: Weak D and Partial D

This is where Rh gets particularly interesting and clinically critical! Not all D+ individuals express the D antigen equally strongly or completely

  • Weak D (Formerly Du)
    • Biochemistry/Genetics: Usually caused by missense mutations (SNPs) in the RHD gene that change single amino acids. These changes often occur in the transmembrane or intracellular regions of the RhD protein
    • Result: Leads to fewer D antigen sites on the RBC surface (a quantitative difference). The D antigens present are generally structurally normal
    • Serology: React weakly or are non-reactive with some anti-D reagents in direct agglutination tests (IS), but usually react (sometimes weakly) at the AHG phase
    • Clinical Significance
      • As Recipients: Most Weak D individuals (especially Types 1, 2, 3 which are common) do not make anti-D if exposed to D+ blood. For safety, they are often managed as D-negative for transfusion
      • As Donors: Weak D blood units are considered D-positive because they can immunize D-negative recipients
    • Molecular testing is often needed to confirm Weak D type
  • Partial D
    • Biochemistry/Genetics: Often caused by gene rearrangements where parts of the RHD gene are replaced with corresponding parts of the RHCE gene (creating hybrid genes), or by mutations causing significant changes in the external loops of the RhD protein
    • Result: The RhD protein is altered, meaning some D epitopes (parts of the antigen recognized by antibodies) are missing (a qualitative difference). The number of antigen sites might be normal or reduced
    • Serology: Reactivity with anti-D reagents can vary from strong positive to weak or negative, depending on which epitopes are missing and the clones used in the anti-D reagent
    • Clinical Significance
      • HIGH RISK: Partial D individuals can make allo-anti-D against the epitope(s) they are missing if exposed to D+ blood (containing the complete D antigen)
      • Management: Partial D individuals must be identified and transfused with D-negative blood. Pregnant Partial D women who are carrying a D+ fetus may need Rh Immune Globulin (RhIG)
    • Distinguishing Weak D from Partial D is crucial and often requires molecular genotyping

Other Important Rh Antigens

While D, C, c, E, e are the main players, others exist:

  • G Antigen: Almost always present on cells that have either D or C antigen (or both). Anti-G reacts like a combination of anti-D + anti-C. Important in obstetric settings
  • f Antigen (ce): Expressed when c and e are present on the same RhCE protein molecule (i.e., inherited in cis, from the same haplotype, like dce)
  • Cw Antigen: An alternative allele to C/c at the C/c locus. Low prevalence
  • V (RH10) and VS (RH20): Low prevalence in Caucasians but higher prevalence in individuals of African descent. Associated with specific RHCE gene variants

Rhnull Syndrome

A rare condition where individuals lack all Rh antigens (D, C, c, E, e, etc.) on their RBCs

  • Genetics
    • Regulator Type: Mutations in the RHAG gene. No functional RhAG means RhD/RhCE proteins cannot get into the membrane, even if RHD/RHCE genes are normal
    • Amorphic Type: Have a deletion of the RHD gene plus mutations in both inherited RHCE alleles, along with a normal RHAG gene
  • Biochemistry: Absence of the Rh protein complex (RhD, RhCE, RhAG)
  • Clinical Features: Associated with red blood cell membrane abnormalities (e.g., stomatocytosis, increased osmotic fragility) leading to mild-to-moderate chronic hemolytic anemia
  • Transfusion: Extremely difficult. These individuals can make antibodies to high-prevalence antigens related to the Rh complex (like anti-Rh29 - total Rh). They need blood from other Rhnull donors, which is exceptionally rare

Key Terms

  • RHD Gene: Codes for the protein carrying the D antigen
  • RHCE Gene: Codes for the protein carrying the C/c and E/e antigens
  • RHAG Gene: Codes for the essential Rh-associated glycoprotein needed for Rh protein expression
  • RhD/RhCE Proteins: Non-glycosylated, 12-pass transmembrane proteins forming the structural basis of Rh antigens
  • RhAG Protein: Glycosylated transmembrane protein essential for RhD/RhCE membrane integration
  • Antithetical Antigens: Antigens coded by alleles at the same locus (e.g., C and c, E and e)
  • Haplotype: A set of linked genes inherited together on one chromosome (e.g., DCe)
  • Weak D: Quantitative reduction in D antigen expression, usually due to mutations affecting protein transport or stability. Cells have fewer D sites
  • Partial D: Qualitative alteration of the D antigen, with missing epitopes, often due to hybrid genes or mutations in external loops. Individuals can make allo-anti-D
  • Epitope: The specific part of an antigen molecule recognized by an antibody
  • Rhnull: Absence of all Rh antigens due to defects in RHAG (regulator) or RHD/RHCE (amorphic). Associated with hemolytic anemia
  • Immunogenicity: The ability of an antigen to provoke an immune response (antibody production). D is highly immunogenic