AHG P.1

• Hemagglutination


  The single most important in vitro immunologic reaction in blood banking because it is the endpoint of almost all systems designed to detect RBC antigens and antibodies
• Hemagglutination reactions


  1. RBC sensitization

  2. Lattice formation
• RBC sensitization


  Combination of single paratope and single epitope in a reversible reaction that follows the law of mass of action and has an associated equilibrium constant. Antigen and antibody are held together by noncovalent attractions.
• Factors affecting RBC sensitization

  • Antibody class (IgM better for agglutination than IgG)
  • Nature of antibody (affinity and avidity)
• Lattice formation


  Multiple RBCs with bound antibody form a stable latticework through antigen-antibody bridges formed between adjacent cells. Basis of all visible agglutination reactions.
• Zeta potential


  The difference in charge at the surface between the inner and outer cation layers, which keeps RBCs in solution about 24nm apart
• Rouleaux formation


  Phenomenon where zeta potential is avoided, causing RBCs to stack like coins. Seen in conditions like multiple myeloma, Waldenstrom's macroglobulinemia, and with plasma expanders.
• Water potential


  The water creates a surface tension that helps to keep the cells apart
• Factors affecting hemagglutination reaction

  • Temperature
  • Incubation
  • pH
  • Ionic strength
  • Antibody concentration
  • Zeta potential
• Grading of hemagglutination reactions


  • Tube testing/conventional method
  • Column agglutination/gel technology
  • Solid-phase technology
• Tube testing/conventional method


  Positive result: one solid agglutinate at the bottom of the tube. Negative result: no/absence of agglutination.
• Column agglutination/gel technology


  Positive result: solid band of agglutination at the top of the tube. Negative result: well delineated pellet at the bottom of the tube.
• Solid-phase technology


  Positive result: diffuse homogenous appearance. Negative result: solid button at the bottom.
• Antiglobulin testing (AHG)


  Also called Coombs test. Principle: antihuman globulin obtained from immunized non-human species bind to human globulins such as IgG or complement, either free in serum or attached to antigens on RBCs.
• There are antibodies that are non-agglutinating (RBCs). IgG and IgM are good.
• Reading for agglutination


  1. Shake tube gently to dislodge cells

  2. Observe for agglutinates as cell button resuspends

  3. Use concave mirror for proper illumination and to differentiate aggregates from free cells
• TLDR: shake, check for agglutinates, if present then positive, if not then negative
• Antiglobulin testing (AHG)


  Also called Coombs test
• Principle: antihuman globulin obtained from immunized non-human species binds to human globulins such as IgG or complement, either free in serum or attached to antigens on RBCs
• Non-agglutinating antibodies


  IgG and IgM are good for agglutination
• IgM


  Does not need AHG reagent to see agglutination, as it has a pentameric (large) structure
• IgG


  Needs AHG reagent to cause agglutination, as it has a monomeric (small) structure
• AHG reagent source


  Mouse (monoclonal) and rabbit (polyclonal), binds to IgG or complement
• Coombs and associates described the use of the antiglobulin test for the detection of weak and nonagglutinating Rh antibodies in serum


  1945
• Coombs and co-workers described the use of AHG to detect in-vivo sensitization of the RBCs of babies suffering from hemolytic disease of the newborn (HDN)

  1946
• Although Coombs and associates were instrumental in introducing the antiglobulin test to blood group serology, the principle of the test had in fact been described by Moreschi in 1908
• Moreschi's studies involved the use of rabbit anti-goat serum to agglutinate rabbit RBCs that were sensitized with low non-agglutinating doses of goat anti-rabbit RBC serum
• Polyspecific antihuman globulin


  Contains antibody to human IgG and to the C3d component of human complement, may also contain other anti-complement antibodies
• Commercially prepared polyspecific AHG contains little, if any, activity against IgA and IgM heavy chains, but may contain antibody activity to kappa and lambda light chains common to all immunoglobulin classes
• Classic/conventional method for producing AHG

  1. Inject human serum or purified globulin into rabbits (sheep, goat) to trigger production of antibody to human serum

  2. Absorb with A1, B, and O cells to remove heterospecific antibodies
• Polyclonal antibodies


  A mixture of antibodies from different plasma cell clones, recognizing different antigenic determinants or the same portion of the antigen but with different affinities
• Monospecific antihuman globulin


  Contains only one antibody specificity, either anti-IgG or antibody to specific complement components
• Anti-IgG


  Contains antibodies specific for the Fc fragment of the gamma heavy chain of the IgG molecule, does not react with IgG4
• Anti-complement


  Contains no activity against human immunoglobulins, reactive against designated complement components only
• Hybridoma technology (monoclonal antibody technique)

  1. Immunize mice with purified human globulin

  2. Fuse mouse spleen cells with myeloma cells to create hybridomas

  3. Screen hybridomas for required antibody specificity and affinity

  4. Propagate selected clones in tissue culture or mice
• Monoclonal antibodies


  All antibody produced by a clone of hybridoma cells are identical in terms of antibody structure and antigen specificity
• Differentiation of polyclonal vs. monoclonal antibodies

  • Polyclonal: Conventional/classical, rabbit, different epitopes, different clones and plasma cells, hetero antibody population, high cross reactivity, inexpensive
  • Monoclonal: Hybridoma technology, mice, specific epitopes, same clones, homo antibody population, low cross reactivity, expensive
• Antibodies required in AHG tests

  • Anti-IgG
  • Anti-complement
• Anti-IgG


  Majority are mixture of IgG1 and IgG3 subclasses, may also contain IgM and rarely IgA
• Anti-complement


  Detects complement components C3 and C4 fixed to RBCs, anti-C3c is most important to avoid non-specific reactions