BB F2

• Antihuman Globulin (AHG)


  Detects non-agglutinating antibody

  
• 2 Types of Antiglobulin Test


  • Direct Antiglobulin Test (DAT)
  • Indirect Antiglobulin Test (IAT)
• Direct Antiglobulin Test (DAT)


  Detects in-vivo sensitization of RBC

  
• Indirect Antiglobulin Test (IAT)


  Detects in-vitro sensitization of the RBC

  
• Indirect Antiglobulin Test (IAT)


  1. Incubation phase

  2. Use of Anti-Human Globulin (AHG)

  
• Hemagglutination


  The single most important in vitro immunologic reaction in blood banking
• Hemagglutination Reactions


  1. RBC Sensitization

  2. Lattice Formation
• RBC Sensitization


  Combination of single paratope and single epitope in a reversible reaction
• Lattice Formation


  Multiple RBCs with bound antibody form a stable latticework through antigen-antibody bridges

  
• Zeta Potential


  Difference in charge at the surface between the inner and outer cation layers, keeps RBCs apart
• Rouleaux Formation


  Phenomenon where zeta potential is being avoided, "stacked of coin appearance"
• Resolution of Rouleaux Formation

  1. Saline Replacement Technique

  2. Water Hydration
• Enhancement Media


  Polyethylene glycol, removes water molecules to increase antigen-antibody reactions
• Factors Affecting Hemagglutination Reaction


  • Temperature
  • Incubation Time
  • pH
  • Ionic Strength
  • Antibody Concentration
  • Zeta Potential
• Tube Testing / Conventional / Traditional Method


  • Positive Result: ONE SOLID agglutinate / aggregate 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 tube
  • Negative Result: a well delineated PELLET at the BOTTOM of the tube
• Solid-Phase Technology


  • Positive Result: Diffuse / homogenous appearance
  • Negative Result: SOLID BUTTON

  
• Hemolysis is considered the strongest positive reaction that can occur and indicates the presence of potent, complement-fixing antibody
• Column agglutination (gel technology)


  Commonly used in the hospital
• Positive results (column agglutination)


  Solid band of agglutination at the top of tube
• Negative results (column agglutination)


  Well delineated pellet at the bottom of the tube
• Solid-phase technology


  Uses microplates
• Positive results (solid-phase technology)


  Diffuse/homogenous appearance

  
• Negative results (solid-phase technology)

  Solid button
• In a microplate, agglutination is diffused, meaning there is a binding of antibody and antigen

  
• In a microplate, antibodies are at the bottom but the antigens did not react to the antibodies. Hence, the unbound red cells will accumulate in the middle

  
• Hemolysis


  Strongest positive reaction that can occur and indicates the presence of potent, complement-fixing antibody
• When there's antigen-antibody reaction, it results to hemolytic reaction

  
• Complements binds to the Fc portion of the antibody
• Reading for agglutination


  1. Shake the tube gently using wrist action until all cells are dislodged

  2. Observe for agglutinates as the cell button begins to resuspend

  3. Use concave mirror to differentiate aggregates from free cells
• Antiglobulin/antihuman-globulin testing (AHG)


  Also called Coombs' test

  
• Principle of AHG


  Antihuman globulins (AHGs) obtained from immunized nonhuman species bind to human globulins such as IgG or complement, either free in serum or attached to antigens on red blood cells (RBCs)

  
• Purpose of AHG


  Detect IgG and/or complement-sensitized RBCs
• IgG antibodies are termed nonagglutinating because their monomer structure is too small to agglutinate sensitized RBCs directly

  
• The addition of AHG containing anti-IgG to RBCs sensitized with IgG antibodies allows for hemagglutination of these sensitized cells

  
• Anti-human globulin reagents


  • Anti-IgG binds to the IgG that is attached to the antigen
  • Anti-C3d binds to the antibodies that is fixed by the complement

  
• Polyspecific/polyclonal antihuman globulin


  • Contains antibody to human IgG and to the C3d component of human complement
  • May also contain other anticomplement antibodies

  
• Commercially prepared polyspecific AHG contains little, if any, activity against IgA and IgM heavy chains
• Classic/conventional method of AHG preparation


  • Involves injecting human serum or purified globulin into rabbits (sheep, goat), and an immune stimulus triggers production of antibody to human serum
  • Antigen is harvested and absorbed with A1, B and O cells to remove heterospecific Ab

  
• Polyclonal antibodies


  A mixture of antibodies from different plasma cell clones that recognize different antigenic determinants (epitopes) or the same portion of the antigen but with different affinities