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.