Antiglobulin Test (AHG) and Hemagglutination

• The Antiglobulin (AHG) test is also referred to as the Anti-human globulin test or Coomb's test
• It is an essential methodology in transfusion medicine
• The two types of antiglobulin tests are:
  • Direct antiglobulin test (DAT), also known as Direct Coomb's test
  • Indirect antiglobulin test (IAT), also known as Indirect Coomb's test
• The test principle involves antihuman globulins obtained from immunized nonhuman species (rabbits, mice)
• These bind to human globulins like IgG (anti-IgG) or complement (anti-C3d), either free in serum or attached to antigens on red blood cells
• The addition of AHG allows hemagglutination of RBCs sensitized by IgG auto/alloantibody or complement
• It detects IgG or complement-sensitized RBCs
• Some IgG antibodies are termed nonagglutinating or incomplete because their single monomer structure is too small to directly agglutinate sensitized RBCs
• IgM antibodies, due to their large pentameric structure, bind to corresponding antigen and directly agglutinate RBCs suspended in saline

History of the Antiglobulin Test

• In 1945, Coombs described the use of the antiglobulin test for detecting weak and non-agglutinating Rh in serum
• In 1946, Coombs and coworkers described using AHG to detect in vivo sensitization of RBCs in babies with hemolytic disease
• Coomb's procedure involved:
  • Injecting human serum into rabbits to produce antihuman serum
  • Adsorption to remove heterospecific antibodies
  • Dilution to avoid prozone (excess antibodies)
  • Ensuring AHG serum retains sufficient antibody activity for cross-linking IgG-sensitized RBCs
• Cross-linking of sensitized RBCs by AHG produces hemagglutination
• Indicates reaction with an antigen on the cell surface
• Early AHG reagents were prepared using a crude globulin fraction as the immunogen
• In 1947, Coombs and Mourant demonstrated that the antibody activity that detected Rh antibodies was associated with the anti-gamma globulin fraction in the reagent
• In 1951, Dacie observed different reaction patterns when diluting AHG when testing cells sensitized with warm versus cold agglutinins
• In 1957, Dacie and coworkers published data that the reactivity of AHG to cells sensitized with warm antibodies resulted from anti-gamma globulin activity
• Anti-nongamma globulin activity was responsible for the activity of cells sensitized by cold antibodies.

Antiglobulin Test Uses

• Used to detect RBCs sensitized with:
  • IgG Alloantibodies
  • IgG Autoantibodies
  • Complement components
• Sensitization can occur in vivo or in vitro
• AHG detects in vitro sensitization of RBCs by using a two way technique referred to as the Indirect antiglobulin test (IAT)
• In vivo sensitization is detected by a one-stage procedure called the Direct antiglobulin test (DAT)

Direct Antiglobulin Test (DAT)

• This test detects in vivo sensitization of RBCs with IgG or complement components, and does not require incubation at 37°C
• The detection level is between 100-500 IgG molecules per RBC and 400-1100 molecules of C3d per RBC
• The clinical applications include:
  • HDN: Maternal antibody coating fetal RBCs
  • HTR: Recipient antibody coating donor RBCs
  • AIHA: Autoantibody coating individual's RBCs

Procedure for the Direct Antiglobulin Test

• Collect a whole blood specimen in EDTA to avoid in vitro complement attachment from refrigerated, clotted samples
• Perform an initial DAT with one drop of 3-5% RCS plus polyspecific AHG reagent with anti-IgG and anti-C3d
• If the test is positive:
  • Perform a DAT panel to detect if IgG or complement components sensitized the RBC
  • Include a saline control to detect spontaneous agglutination without AHG reagent
• DAT panel: monospecific anti-IgG and anti-C3d reagents
• Interpret the DAT Panel Patterns of Reactivity in Autoimmune Hemolytic Anemia

In Vivo Phenomena Associated With a Positive DAT

• Transfusion:
  • Recipient alloantibody and donor antigen can cause alloantibodies in the recipient to react resulting in a recent transfusion
  • Donor antibody and recipient antigen present in donor plasma can react with antigen on a transfusion recipient's RBCs
• Drug-Induced:
  • Type I (hapten-dependent Ab): Drug covalently binds to membrane proteins, stimulating hapten-dependent Ab
  • Type II (autoantibody): Drug induces autoantibody specific for RBC proteins
  • Type III (drug-dependent Ab): Drug induces Ab that binds to RBC only when drug is present in soluble form
• Autoimmune Hemolytic Anemia:
  • WAIHA (IgG and/or C3): Autoantibody reacts with patient's RBCs in vivo
  • CAS (C3): IgM binds complement and returns with warmer parts of circulation
  • PCH (IgG): IgG reacts with RBC's in colder parts and causes complement to irreversibly bind to RBCs
• Hemolytic disease of fetus and newborn:
  • Maternal alloantibody crosses placenta and coats fetal RBCs
• Miscellaneous issues:
  • Absorbed proteins and administration of equine preparations can coat recipient's RBCs
  • Nonantibody-mediated binding of immunoglobulin in patients with hypergammaglobulinemia

Indirect Antiglobulin Test (IAT)

• The test detects in vitro sensitization of RBCs using IgG or complement components at a 37°C incubation
• For a positive reaction to occur, there must be between 100 and 200 IgG molecules on the cell
• The tests include:
  • Antibody detection uses compatibility testing by showing the recipient antibody reacting with donor cells
  • Antibody identification uses antibody screening by reacting with screening cells
  • Antibody titration uses Rh antibody titer
  • RBC phenotype used to detect anti-sera using RBCs

Procedure for Indirect Antiglobulin Test

• Incubate patient's RBCs with antisera to allow time for antibody attachment to the RBC antigen
• Perform a minimum of three washings to remove free globin molecules not attached to the antigen
• Add the AHG reagent to bridge or link sensitized RBCs for visible hemeagglutination
• Includes RBC antigen + antibody + anti-IgG
• Then centrifuge to accelerate agglutination
• Examine for agglutination and grade the reactions
• If the result is negative:
  • Add O Check cells/Coomb's control cells (Group O, D positive RBCs coated with anti-D (IgG)); negative reactions should demonstrate hemeagglutination
  • Checks for neutralization of AHG reagent
  • If still negative after addition of check cells, the test is invalid, and you must repeat procedure

Antiglobulin (AHG) Reagents

• Antihuman globulin reagents can either be polyspecific or monospecific
• Polyspecific AHG contains antibody to human IgG (anti-IgG) and to the C3d component of human complement (anti-C3d)
• Includes Anti-C3b, Anti-C4b, and Anti-C4d other anti-complement components
• Include antibody activity to kappa and lambda light chains common to all immunoglobulin classes, thus reacting with IgA or IgM molecules
• Monospecific contains only one antibody specificity, either anti-IgG or antibody to specific complement components such as C3b or C3d
• Anti-IgG contain no activity for anti-complement
• Their reagents contain antibodies specific for the Fc fragment of the gamma heavy chain
• Anti-complement reagents are against only the complement, with no activity for human immunoglobulins
• The preparation methods for AHG reagents:
  • Classic method
  • Hybridoma technology

Classic Method (Hyperimmunization of Rabbits)

• This method produces polyclonal antibodies from different plasma clones that recognize different antigenic determinants/epitopes
• The process injects human serum or purified globulin into laboratory animals like rabbits
• Human IgG injected into a rabbit for anti-IgG production
• Human complement components injected into a rabbit for anti-complement
• Polling of anti-IgG from many immunized rabbits are essential for different IgG antibodies that are detecting for reagents in routine use.
• Polyspecific AHG starts with the purification of the immunogen from a large pool of normal sera
• Conventional polyspecific antiglobulin reagents are producing an anti-globulin with a human antigen

Hybridoma Technology

• This is also known as Kohler and Milstein technique
• It produces monoclonal antibodies that derive from one plasma and recognized a single epitiope
• The procedure involves:
• Immunizing mice with purified human globulin
• Fusing mouse spleen cells with antibody-secreting lymphocytes with myeloma cells
• Screening hybridomas for antibodies with the specificity and affinity required
• Propagating antibody-secreting in tissue culture with HAT medium or into mice harvested as ascites

Factors that Affect the Test

• These include:
  • Ratio of serum to cells
  • Reaction medium
  • Temperature and incubation time
  • RBC washing
  • AHG addition
  • Centrifugation for reading
• An increasing ration of cells increases reactivity, a 40:1 ration is the minimum. and for weak antibodies it's 133:1
• Medium - Macromolecules of allow antibody coated cells to come into closer contact with each other, or using PEG
• Temperature: 37°C for IgG antibodies and complement activation
• Washing of requires a 3 minimum washings
• Saline should have a high pH so there is no false positives
• AHG added is to minimize chance of being neutralized, then centrifuged for 20 seconds.

Sources of Testing Error

• The most common errors for positives is because of inadequate washing, contaminated AHG, and improper addition
• For negatives, it include high protein, cold temperature and testing interruption.

Explore the principles behind the Antiglobulin Test (AHG) and hemagglutination assays, including the role of IgG antibodies and the significance of Coombs' work. Learn about the importance of cell washing, saline quality, and the purpose of adsorption in AHG reagent preparation. Understand the mechanism of action of AHG reagent.