Serological endpoints: Agglutination - Coombs Testing
As previously discussed, anti-human globulin (AHG) contains an IgG with specificity for the constant region of other IgG antibodies. When this binding occurs, a bridge is created between IgG-bound red cells, causing them to form visible agglutinates.
Since the binding of AHG requires warming the sample to 37C, AHG cannot be used to directly detect the binding of IgM antibodies, which tend to elute off the surface of red cells at that temperature. However, since IgM antibodies are potent activators of complement, their binding to the red cell surface can be inferred by the detection of residual complement split products (typically C3b or C3d). AHG reagents that contain a mixture of anti-IgG and anti-C3 are called polyspecific, whereas those that contain only one or the other are called monopecific. Polyspecific testing is usually performed first, and if positive is followed by monospecific testing.
AHG testing can be used to detect the presence of anti-red cell antibodies either directly or indirectly, depending on what type of information is being sought.
- Direct: AHG is used to detect antibodies which have already bound to the red cells (typically through in vivo sensitization). Direct testing can detect between 100 to 500 molecules of IgG per red cell, or 400 to 1000 molecules of C3 per red cell. The question being asked by a direct AHG test is: "Have antibodies bound to the surface of this red cell?", and is performed when investigating for autoimmune hemolytic anemia, delayed hemolytic transfusion reactions or hemolytic disease of the fetus or newborn
- Indirect: AHG is used to detect antibodies in a plasma sample. In this instance, plasma is first incubated with red cells (in vitro sensitization) and then AHG is added to detect agglutination. Indirect testing can detect between 100 to 200 IgG or C3 molecules per red cell. The question being asked by an indirect AHG test is: “Does this plasma contain antibodies to red blood cells?” and is performed during an antibody screen or investigation, or when crossmatching red blood cells. Indirect AHG testing is also used when phenotyping red cells: a reagent containing an IgG antibody against a specific antigen such as C or Kell is first incubated with RBCs and then AHG is added to detect agglutination. However, the majority of phenotyping reagents are now IgM, which are directly agglutinating and no longer require the use of AHG.
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