AHG

• Coombs’ test or AHG test was developed by Coombs, Mourant, and Race in 1945 for the detection of weak and non-agglutinating Rh antibodies
• Principle of AHG test:

  • Antihuman globulins obtained from immunized nonhuman species bind to human globulins such as IgG or complement (C3, C4), either free in serum or attached to antigens on red blood cells (RBCs)
  • Can be monospecific (antihuman globulins only) or polyspecific containing anti-complement component
  • Detection method: hemagglutination
• AHG reagent containing anti-IgG to RBCs sensitized with IgG antibodies allows for hemagglutination of these sensitized cells
• Antiglobulin Test:

  • Direct Antiglobulin Test (DAT) detects in vivo sensitization of RBCs with IgG or complement components
  • Clinical conditions resulting in in vivo sensitization include Hemolytic disease of the fetus and newborn (HDFN), Hemolytic transfusion reaction (HTR), Autoimmune and drug-induced hemolytic anemia (AIHA)
• Direct Antiglobulin Test (DAT) Panel:

  • Initial test: one drop of a 3% to 5% suspension of washed RBCs + polyspecific (anti-IgG, anti-C3d) reagent
  • If positive, proceed to testing using monospecific reagent (anti-IgG or -C3d)
  • Some laboratories run polyspecific and monospecific reagents at one time as well as a saline control to detect spontaneous agglutination of cells or reactions occurring without the antigen
• The antiglobulin test is used to detect RBCs sensitized by IgG alloantibodies, IgG autoantibodies, and complement components
• AHG reagents containing anti-IgG are needed for the detection of IgG antibodies because the IgG monomeric structure is too small to directly agglutinate sensitized RBCs
• Polyspecific AHG sera contain antibodies to human IgG and the C3d component of human complement
• Monospecific AHG sera contain only one antibody specificity: either anti-IgG or antibody to anti–C3b–C3d
• Classic AHG sera (polyclonal) are prepared by injecting human globulins into rabbits, and an immune stimulus triggers production of antibody to human serum
• Hybridoma technology is used to produce monoclonal antiglobulin serum
• The DAT detects in vivo sensitization of RBCs with IgG or complement components. Clinical conditions that can result in a positive DAT include HDFN, HTR, and AIHA
• The IAT detects in vitro sensitization of RBCs and can be applied to compatibility testing, antibody screen, antibody identification, RBC phenotyping, and titration studies
• A positive DAT is followed by a DAT panel using monospecific anti-IgG and anti-C3d to determine the specific type of protein sensitizing the RBC
• There are multiple sources of error that can be introduced into the AHG procedure
• Incubate RBCs with antisera:Allows time for antibody molecule attachment to RBC antigen
• Perform a minimum of three saline washes: Removes free globulin molecules
• Add antiglobulin reagent: Forms RBC agglutinates (RBC Ag + Ab + anti-IgG)
• Centrifuge: Accelerates agglutination by bringing cells closer together
• Add antibody-coated RBCs to negative reactions: Checks for neutralization of anti - sera by free globulin molecules
• Ratio of serum to cells: Increasing the ratio of serum to cells increases the sensitivity of the test system.
• Reaction medium: Albumin, LISS Polythylene glycol
• Temperature: optimal at 37°C
• Incubation time: , incubation times may vary between 30 and 120 minutes
• Washing of RBC: removes free unbound serum globulins
• Saline for washing: should be fresh
• Addition of AHG: minimize the chance of antibody eluting
• False Positive

  •   Improper specimen (refrigerated, clotted) may cause in vitro complement attachment