Genetics and Immunology - Blood Grouping and MHC Typing Notes

Blood Grouping and MHC Typing

History of Blood Transfusions

  • Large blood losses have serious consequences (15-30% = weakness, Over 30% = shock).
  • Transfusions must be of the same blood group.
  • 1600s: Discovery of blood circulation by William Harvey; first recorded blood transfusion in dogs.
  • 1800s: James Blundell completes first successful human blood transfusion; milk and saline infusions used as blood replacements.
  • 1900s: Discovery of ABO blood groups (Karl Landsteiner); development of cross-matching and indirect transfusions.
  • 2000s: Introduction of Blood Safety and Quality Regulations (BSQR); routine use of automation and computerized systems.
  • Indications for blood transfusion: Medical conditions (anemia, cancer, bleeding disorders) 64%, Surgery (Planned and Emergency surgery , Trauma) 25%, Blood loss following Childbirth 11%.

Blood Group Antigens

  • Located within RBC membrane structure and genetically coded.
  • Variation doesn't normally affect red cell membrane function.
  • Composed of lipid, carbohydrate, and/or protein molecules.
  • Over 400 different systems, but ABO and Rhesus are most important.

Inheritance of ABO Blood Groups

  • One allele inherited from each parent, resulting in 6 possible genotypes but only 4 phenotypes.
  • A antigens: AA or AO.
  • B antigens: BB or BO.
  • AB antigens: AB.
  • O antigens: OO.

Antigens – A, B and H

  • The 3 antigens of the ABO system are A, B, and H (not O).
  • H gene locus on Chromosome 19, independent of the ABO locus. Enzymatic addition of L-Fucose, and A and B genes code for enzymatic additions to the H antigen.
  • Addition of N-acetyl-D-Galactosamine results in A Antigen, and addition of D-galactose results in B Antigen.
  • Group O expresses the H antigen only.

ABO Antibodies

  • Naturally occurring, developed from approximately 4 months of age due to environmental stimulation.
  • Governed by Landsteiner’s Law: Antigen present; Antibody Absent. Antigen Absent; Antibody Present.

Bombay Blood Group

  • Absence of H gene (hh) results in no production of H antigen.
  • A and B genes can be present but cannot add antigens, abbreviated to Oh.
  • Circulating antibodies to A, B, and the H antigen.
  • Unable to accept conventional Group O; very rare.

The D Antigen

  • Part of the RH (Rhesus) Blood Group System, gene locus on chromosome 1.
  • D antigen is a major carrier protein; D is dominant allele over d.
  • d does not have a gene product, resulting in D Positive or D Negative.
  • 77% of donors are D positive, 23% are D negative.

Importance of D Antigen

  • Highly antigenic and immunogenic, able to stimulate antibody production.
  • 60% of D Neg patients transfused with D Pos blood develop Anti-D.
  • Antibody develops due to sensitization by pregnancy or transfusion.
  • Two genes on Chromosome 1: D gene and CE gene.

RH Genotypes

  • D and CE gene are inherited independently.
  • D has no corresponding d antigen; however, both C & E have antithetical antigens.
  • The 5 main Rh antigens on red cells – C, c, D, E, e.

Rh Dangers During Pregnancy

  • Mismatch of an Rh– mother carrying an Rh+ baby can cause problems (Hemolytic disease of the newborn).
  • The immune system is sensitized after the first pregnancy.
  • In a second pregnancy, the mother’s immune system produces antibodies to attack the Rh+ blood.

ABO Compatibility

  • Shows which blood groups can be given to which blood group.

Other Blood Groups

  • Over 400 described, but only approximately 15 clinically significant.
  • Exposure via transfusion or pregnancy may result in antibody production.
  • Must screen for the presence of antibodies and obtain antigen-negative blood for transfusion.

ABO/D Typing

  • Traditionally performed on a slide or tile; nowadays use either a tube (manual) or column agglutination card (automated).
  • Forward Group: Known anti-sera for a specific antigen (e.g., A, B, D) is mixed with patient’s red cells. Agglutination means the antigen is present; non-agglutination means it is absent.
  • Reverse Group: Known Red cells (A or B) is mixed with patient’s plasma. Agglutination means the antibody is present; non-agglutination means it is absent.
  • A check of forward group!

Tube Test and Column Agglutination Technology

  • Tube Test: Reaction performed in a test tube; more standardized than tile. Centrifugation forms a cell button. Dispersal indicates agglutination (Positive) or not (Negative).
  • Column Agglutination Technology: Gel beads with anti-sera attached. Centrifuge; red cells with antigen stay at top (Positive); red cells without antigen pass through (Negative).

IgM vs IgG

  • IgM antibodies: Large pentameric antibody, causes direct agglutination, causes intravascular hemolysis by binding complement, cannot cross the placenta, and typically ‘cold’ acting. ABO antibodies are IgM.
  • IgG antibodies: Small antibody (2 binding sites), cannot cause agglutination, induces complement destruction via macrophages, crosses the placenta, and typically active only at 37°C. RH and most other antibodies.

Indirect Antiglobulin Test (IAT)

  • ABO antibodies are frequently IgM; however, most other antibodies are IgG. IgG antibodies CANNOT cause agglutination alone.
  • Addition of a reagent to act as a bridge: Anti-human globulin (AHG), sometimes called Coombs reagent.
    1. Plasma containing IgG Antibodies
    2. Addition of Red cells with corresponding antigen
    3. Antibodies bind to antigen
    4. Addition of AHG
    5. AHG crosslinks IgG antibodies - Agglutination

The Use of IAT Tests

  • Antibody screen uses reagent cells with known antigen expression to pre-select antigen-negative units.
  • Crossmatch uses donor cells with unknown antigen expression to check for potential errors and ABO incompatibility.

Crossmatch

  • Final pre-transfusion test to check compatibility of patient’s plasma with prospective donor unit.
  • IAT test: Recipient's Plasma VS Donor RBCs.
  • Results: No agglutination – Compatible; Agglutination – Incompatible.

Tissue Typing

  • Determine the histocompatibility antigens of both recipient and potential donor and use the organ with the fewest mismatches.

Classification of the Grafts

  • Autograft: Within an individual.
  • Isograft: use of tissue from an identical twin
  • Allograft: same species
  • Xenograft: Between species.

Effectors of Rejection

  • Major Players: T Cells, B cells, Antigen-presenting cells, MHC (Most Important).

HLA and MHC

  • HLA = Human Leucocyte Antigen system which forms part of the Major Histocompatibility Complex (MHC).
  • Found on the short arm of chromosome 6.
  • MHC antigens are integral to the normal functioning of the immune response.
  • Every person inherits each of the following antigens from each parent: HLA-A antigen, HLA-B antigen, HLA-Cw antigen, HLA-DR antigen, HLA-DQ antigen, and HLA-DP antigen.

Typing Methods

  • SEROLOGY: was the ‘historical gold’ standard but Now being superseded by molecular techniques.
  • CELLULAR rarely used now. Originally used for Class II typing.
  • MOLECULAR with polymerase chain reaction (PCR).
    • PCR with sequence-specific primers (PCR-SSP)
    • PCR using sequence -specific oligonucleotide probes (PCR- SSOP)
    • PCR-sequence based typing (PCR-SBT)

Crossmatch Test

  • After tissue typing and antibody screening are complete and a potential donor has been identified, the final test is called a crossmatch test.
  • Complement-dependent cytotoxic (CDC) crossmatching.
  • Patients transplanted against a positive IgG CDC cross-match are at high risk of hyperacute rejection.
  • The test detects both HLA-specific and non-HLA complement-fixing IgG and IgM antibodies.

Crossmatch Flow Cytometric Crossmatch (FCXM)

  • FCXM is a sensitive method for detecting donor-specific HLA and non-HLA antibodies, identifying both complement-fixing and non-complement-fixing types.
  • It uses donor lymphocytes, sorted by fluorescent markers, and patient serum, with anti-IgG labeling to detect bound antibodies via flow cytometry.

Crossmatch Solid Phase Assay Crossmatching

  • Solid-phase assay crossmatching, which isolates HLA antigens on synthetic beads, improves specificity by attributing reactivity solely to HLA antibodies.

Crossmatch Virtual Crossmatching

  • Virtual crossmatching emerged initially in cardiothoracic transplantation due to limited organ ischemia time.
  • This method uses detailed HLA antibody screening and precise donor HLA typing for a pre-transplant paper-based crossmatch.
  • For some cases, CDC or FCXM crossmatching might still be needed, potentially reducing available donor matches.

Crossmatch Test

  • A small amount of the potential donor’s white cells is mixed with a small amount of the recipient’s serum where By exposing the donor’s HLA to the recipient’s serum can determine whether transplantation can be performed.