Coomb's test

Coomb's reagent

• Coomb's reagent (aka antiglobulin serum)
  * Contains polyspecific rabbit or sheep IgG antibodies which are directed against human IgG antibodies (i.e. anti-human-IgG IgG antibodies)
• Anti-human globulin (AHG)
  * Produced in animals after injection of human globulin, complement, or specific immunoglobulin
  * May contain polyspecific antibodies or monospecific
  * ??Coomb's reagent is a subtype of AHG

Direct Coomb's test

• Aim
  = To detect antibody or complement on the RBC that have been sensitized in the patient's body
• Patient's RBC are washed first
• Then AHG is added to the washed cells
• If agglutination occurs
  --> Positive direct Coomb's test
  --> Patient has been sensitised (in vivo) to some antigen on the RBC (i.e. producing antibodies against the RBC cells)

Direct Coomb's test is positive in

• Haemoytic disease (Rh) of the newborn
• Autoimmune haemolytic anaemia
• Drug-induced immune haemolysis
• Haemolytic transfusion reactions

Indirect Coomb's test

• A two stage test
  * Incubation, then add reagent
  * Direct Coomb's does not require incubation
• Aim 
  = To detect the presence of any IgG antibodies (in recipient's plasma) directed against donor's RBC membrane antigen
• These (incomplete) antibodies are not able to cause agglutination by themselves during other testing procedures but may cause problem when transfused

Step 1: Incubation

Donor's RBCs are incubated in recipient's serum at 37C
--> Allow any IgG antibodies (from the recipient) to bind to the corresponding antigen on donor RBC
* i.e. First stage

Step 2: Washing

Removes all serum and unbound IgG in the serum
--> IgG antibodies bound to donor RBC remains

Step 3: Testing with Coomb's reagent

Coomb's reagent is added to the washed RBC

If there are IgG antibodies (from recipient's serum) bound to donor RBC
--> Anti-human-IgG IgG antibodies will bind to the IgG on two RBCs
--> Join them together
--> Agglutination
--> Positive Coomb's test

If no IgG are bound to donor RBC
--> No agglutination
--> Negative Coomb's test

Step 4: Coomb's control test

Coomb's control cells
* RBC known to be coated with IgG

Coomb's control cells are added to the negative Coomb's test
--> Will definitely cause agglutination if the reagent was active

If no agglutination when Coomb's control cells are added
--> Reagent was inactive

Indirect Coomb's test is positive when

• Recipient has antibody in serum against donor RBC (during crossmatching)
• Atypical blood group antibodies (during antibody screening)
• Blood group antibodies in pregnant women
• Serum antibodies in autoimmune haemolytic anaemia