Equis ISSN 2398-2977

Indirect Coombs test

Synonym(s): Indirect antiglobulin test

Contributor(s): Graham Munroe, Prof Michael J Day


  • Detects the presence of circulating anti-RBC antibodies in serum of horses with immune-mediated hemolytic anemia (IMHA).
  • Serum from the affected individual is incubated with normal erythrocytes of known antigenicity, ie a compatible blood group, and the sample monitored for gross agglutination. An equine polyvalent Coombs reagent is then used to enhance detection of any reactivity. If it occurs it is a positive test.
  • It is a test rarely used in veterinary medicine and is generally not available.


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  • Indirect Coombs testing may be undertaken using test tubes or microtitration plates.
  • The tube technique is described below.
  • Two drops of patient serum are placed into appropriately labeled tubes.
  • One drop of washed 2-5% saline-suspended normal RBCs is places into each tube and mixed.
  • The RBCs are allowed to settle or the tubes are centrifuged and then observed for evidence of hemolysis and/or agglutination.
  • The contents of the tubes are then resuspended and the tubes are incubated at 37°C for 30-60 min.
  • The tubes are observed again for signs of hemolysis and agglutination.
  • The cells are washed 3-4 times with saline and the final wash is completely decanted.
  • Antiglobulin reagent (anti-horse polyvalent Coombs reagent) is immediately added in the amount and dilutions as per the manufacturer's guidelines. The contents in the tubes are mixed thoroughly.
  • Allow to settle or centrifuge the tubes and observe again for macroscopic and microscopic agglutination which is positive for the indirect Coombs test.
  • A negative control with saline instead of patient serum would be included throughout.
  • Double-dilutions of the serum from the patient can be carried out (in a microtitration system) and similarly tested. This will allow the maximum dilution of the patient serum that will cause agglutination of the RBCs to be determined and therefore the concentration of the antibody present in the serum to be calculated.


  • Usually only carried out in larger commercial and institutional laboratories.
  • Contact the laboratory before sending the sample.

Technician (extrinsic) limitations

  • The test needs to be carried out with care and professionalism.

Result Data

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Further Reading


Refereed papers

  • Recent references from PubMed and VetMedResource.
  • Wardrop K J (2005) The Coombs test in veterinary medicine: past, present, future. Vet Clin Pathol 34 (4), 325-334 PubMed.