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IHBB-MIDTERMS_LEC_AgAb-Reactions

Properties that Influence Binding

  • Avidity

    • Overall strength of binding between antibody and antigen.

    • Goodness of fit between antibody and its specific epitopes affects reaction strength and rate.

    • Influencing factors: Size, shape, charge of an antigen.

  • Affinity

    • Strength of binding between a single antibody and an epitope of an antigen.

  • Ag-Ab Complex

    • Combination of antigen and antibody creates an immune complex.

Lock-and-Key Concept

  • Red Cell Antigen Characteristics

    • Located on red cells, part of or protruding from the cell membrane.

    • Antibodies are in plasma or serum.

Forces Binding Antigen to Antibody

  • Electrostatic Forces

    • Ionic bonding due to attraction between oppositely charged regions of molecules.

  • Hydrogen Bonding

    • Attraction involving hydrogen atoms between negatively charged groups.

  • Hydrophobic Bonding

    • Weak bonds from the exclusion of water molecules.

  • Van der Waals Forces

    • Attraction from the electron cloud of one atom to the protons of another.

Red Cell Antigens

  • 36 blood group systems with over 250 unique red cell antigens.

  • Blood group systems are based on inheritance patterns of blood group genes.

  • Largest systems assigned by the International Society of Blood Transfusion (ISBT).

  • Some antigens are more immunogenic and must be matched during transfusions (e.g., D negative patients require D negative blood).

Red Cell Antibodies

  • Significant immunoglobulins in transfusion: IgG and IgM.

  • React at body temperature (37°C), with IgG capable of causing immune destruction of transfused red cells, leading to transfusion reactions, anemia, and HDFN.

Antigen-Antibody Reactions

  • ABO Antibodies

    • Primarily IgM class, reactive at room temperature in vitro and 37°C in vivo.

    • Wrong ABO transfusion activates complement system causing hemolysis.

Antigen-Antibody Reactions In Vivo

  • Antibody Screening Test

    • Performed pre-transfusion to detect unexpected antibodies (non-ABO systems).

    • Abs against clinically significant non-ABO blood groups: Duffy, Kell, Kidd, MNS, P, certain Rh types.

  • Naturally Occurring Antibodies

    • Anti-A and anti-B are RBC antibodies found in normal serum/plasma; all other are unexpected.

    • Types:

      • Alloantibodies: To antigens an individual lacks.

      • Autoantibodies: To an individual’s own antigens.

Transfusion, Pregnancy, and Immune Response

  • Alloantibodies

    • Produced in response to transfusions or pregnancy, leading to subsequent sensitization and antibody production.

Complement Proteins

  • Group of serum proteins involved in antigen clearance, cell lysis, and vasodilation.

  • Exist in inactive state; activated into enzymes enhancing immunologic processes.

  • Classical Pathway: Activated by antigen-antibody complexes.

  • Alternative Pathway: Independent of specific antibody activation; initiated by foreign cell-surface constituents.

Biological Effects Mediated by Complement Proteins

  • Anaphylatoxins: C3a, C4a, C5a products causing smooth muscle contraction and increased vascular permeability.

  • Vasoactive Amines: Released by mast cells, basophils, and platelets affecting endothelial cells.

  • Opsonization: Enhances phagocytosis.

  • Phagocytic Cells: Cells like neutrophils and macrophages that engulf foreign particles.

  • Receptors: Surface molecules with high affinity for specific molecules.

Clearance of Antigen-Antibody Complexes

  • Mediated by phagocytic cells (neutrophils, macrophages).

  • Mononuclear phagocyte system efficiently removes Ag-Ab complexes from circulation, acting as a biological filter.

Antigen-Antibody Reactions In Vitro

  • Agglutination

    • Positive reaction indicates successful Ag-Ab interaction.

    • No agglutination indicates a negative reaction.

Serum-to-Cell Ratio

  • Ratio of antigen on red cell to antibody in serum affects agglutination.

Factors Influencing Agglutination Reactions

  • Concentration of Reactants: Antigen and antibody concentrations matter.

  • Centrifugation: Enhances reactions via increased gravitational forces.

  • pH, Temperature, Incubation Time, Ionic Strength also influence reactions.

Factors Influencing the First Stage of Agglutination

  • Temperature: Optimal at 37°C for IgG antibodies.

  • Incubation Time: Ensures Ag-Ab equilibrium.

  • pH: Optimal range of 6.5-7.5 for hemagglutination.

Factors Influencing the Second Stage of Agglutination

  • Antihuman Globulin Reagents:

    • Direct AHG: Detects RBCs coated with antibody/complement.

    • Indirect AHG: Needs incubation with plasma prior.

  • Hemolysis: Indicator of Ag-Ab reactions; visible post-centrifugation.

History of Antiglobulin Test (AGT)

  • Described by Coombs in 1945 for weak/non-agglutinating Rh antibodies.

  • Monospecific and polyspecific AHG reagents derived from animal immunization.

Reagents and Quality Control in AGT

  • Polyspecific AHG Reagents: Pool of various anti-human antibodies.

  • Monospecific AHG Reagents: Contain one antibody specificity.

  • Hybridoma Cells: Created from mouse plasma and myeloma cells for antibody production.

Sources of Error in Antiglobulin Testing

  • False Positive Results: Occur from prior agglutination or improper handling (e.g., dirty glassware).

  • False Negative Results: May arise from improper washing or delay in adding AHG.

Check Cells (Coomb's Negative Control)

  • Commercially prepared red cells with IgG attached for quality control in tests.