AHG AND AB IDENTIFICATION

• Antihuman globulin test


  A technique for detecting cell-bound immunoglobulin
• Role of AHG


  Links/connects cell-bound IgG antibodies
• Differences between IgM and IgG

  • IgM: Pentamer, fastest, first to arrive, leaves fast, naturally occurring, complete Ab, cold-reacting
  • IgG: Monomer, slow-reacting, immune Ab, incomplete Ab, purpose is to coat cells, warm-reacting
• IgG is clinically significant in transfusion, transplantation, and pregnancy
• Stages of antigen-antibody reaction

  1. Sensitization: Antibody recognizes antigen, no agglutination

  2. Agglutination: Antibodies form cross-linkages between cells, visible clumping
• AHG


  Bridging reagent that binds to the Fc region of human IgG to produce a visible reaction
• AHG reagents


  • Polyspecific (broad spectrum): Contains Ab against Ab and complement
  • Monospecific: Contains only one antibody specificity, either anti-IgG or anti-complement
• Direct antiglobulin test (DAT)


  In vivo detection of cell-bound antibodies
• Indirect antiglobulin test (IAT)

  In vitro sensitization of cells with suspected antibodies

  
• Applications of IAT


  • Ab detection (crossmatching, screening, identification)
  • Ab titration
  • RBC phenotyping
• Steps of IAT


  1. Incubate at 37°C

  2. Add AHG (agglutination = lattice formation)
• Delayed AHG reagent addition

  Leads to false negative results due to spontaneous elution of antibodies from the antigen
• Low ionic polybrene technique


  Rapid sensitization of IgG to RBCs and promotion of lattice formation
• Enzyme linked antiglobulin test

  RBC suspension + sensitized IgG + AHG labelled with enzyme + substrate = color reaction
• Sources of error in AHG tests

  • False positive: Autoagglutinable cells, bacterial contamination, polyagglutinable cells
  • False negative: Inadequate washing, AHG reagent deterioration, no AHG addition, zonal phenomenon, poor reading technique
• Factors affecting AHG


  • Temperature, ratio of serum to cells, incubation time, reaction medium, washing of cells, saline used, addition of AHG, centrifugation
• Antibody screening is used to detect clinically significant IgG antibodies outside the ABO blood group system
• Types of antibodies detected in antibody screening

  • Alloantibodies (patient's/donor's serum + screening cells)
  • Autoantibodies (patient's serum + own RBCs)

  
• Screening O cells


  Contain all red cell antigens that would react with antibodies present in the patient's serum

  
• Methods of antibody screening

  • Traditional tube method
  • Gel technology

  
• Phases of antibody screening

  1. Immediate spin: Detects cold IgM antibodies

  2. 37°C incubation with enhancement: Detects warm IgG antibodies

  3. Antiglobulin phase: Detects non-agglutinating IgG antibodies
• Enhancement reagents


  • 22% Albumin, Low ionic strength saline (LISS), Polyethylene glycol (PEG)
• Interpretation of antibody screening results

  • Single alloantibody, Multiple alloantibodies, IgG antibodies, Single antibody with dosage effect, Multiple IgG antibodies, IgM antibody, Warm and cold antibodies, Warm autoantibody

  
• Gel test technology


  Innovative approach to red cell serology that traps agglutinates formed when antigen reacts with antibody

  
• Gel matrix


  Highly sensitive sieve that traps agglutinates based on size exclusion chromatography

  
• Unagglutinated cells settle at the bottom, hemolyzed cells will not be detected by the gel matrix

  
• Dextran

  Used as a sieve, traps agglutinates formed after Ag-Ab reaction
• Dextran (polyacrylamide gel)


  Used as a sieve, provides prolonged contact with the RBC during centrifugation
• Size Exclusion Chromatography


  1. Larger agglutinates formed = higher location in the gel matrix, will not undergo sieving

  2. Unagglutinated = settle at the bottom of the tubes/cell button (pellet), no Ag-Ab reaction

  3. Hemolyzed = will not be reported/detected by the gel matrix, false negative result

  
• Gel Test Card


  • Composed of 6 microtubes (5 x 7 cm)
  • Incubated - sensitization → centrifugation
  • Reaction filter will sieve

  
• Procedure of Gel Test


  1. Addition of cells (patient cell or screening cell)

  2. Addition of plasma/serum (from patient or donor)

  3. Incubation (30 minutes - 1 hour, sensitization occurs)

  4. Centrifugation

  5. Results reporting according to agglutination reaction

  
• Agglutination Reaction Results

  • 4+ Solid band of RBCs at the top of the gel
  • 3+ Agglutinated RBCs in the upper half
  • 2+ RBC agglutinates through length
  • 1+ Agglutination of RBCs in the lower half of the gel column
  • Negative RBC pellets at the bottom
• Advantages of Gel Test

  • Standardization
  • Well-defined and stable results up to 3 days
  • Decreased volume of sample needed (microsampling)
  • Cell washing is no longer performed
  • Enhanced sensitivity and specificity
  • Minimizes contamination and sources of errors compared to traditional tube method

  
• Disadvantages of Gel Test

  • Sample restrictions
  • Need for special equipment
  • Hemolyzed, icteric, lipemic, and other samples suspected with agglutination and rouleaux formation are NOT allowed

  
• Solid-Phase Red Cell Adherence (SPRCA)

  • Developed commercially for the detection of RBC and platelet-related antibodies
  • The ability of plastics, such as polystyrene to absorb proteins from solutions and bind them irreversibly made SPRCA possible
  • If test plasma contains antibodies to antigen = attach to the fixed antigen
  • If the test plasma contains NO antibodies to the antigen = NO attachment to the fixed antigen
  • Solid phase reactions are stable for observation or review for 2 DAYS

  
• SPRCA Procedure


  1. Patient serum or plasma is incubated with intact platelets to allow antibody (if present) to bind to the platelet glycoproteins

  2. Patient serum or plasma is added to microwells that are coated with platelet glycoproteins, allowing antibody (if present) to bind

  3. Unbound antibodies are washed away
• Solid-Phase ELISA Test (MACE®)


  Used primarily for compatibility testing
• MACE® Tests


  Patient serum or plasma is incubated with intact platelets to allow antibody (if present) to bind to the platelet glycoproteins

  
• PAK® Tests


  Patient serum or plasma is added to microwells that are coated with platelet glycoproteins, allowing antibody (if present) to bind

  
• Solid-Phase ELISA Principle


  1. After 30 minutes of incubation period, the reaction is stopped by a sodium hydroxide (NaOH) solution

  2. Finally, the optical density of the color that develops is measured in a spectrophotometer