Equis ISSN 2398-2977

Direct Coombs test

Synonym(s): Direct antiglobulin test, DAT

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


  • Used to confirm the diagnosis of immune-mediated hemolytic anemia Anemia: auto-immune hemolytic (AIHA), but a negative test does not rule out this diagnosis.
  • A direct Coomb's test detects antibody (immunoglobulins) and/or complement attached to the surface of circulating red blood cells (erythrocytes) after the addition of equine Coomb's reagent:
    • The equine Coomb's reagent is a polyvalent antiserum (normally raised in a rabbit or goat) that bind to horse IgG, IgM or complement C3, but does not differentiate between these immunoreactants.
    • A positive test leads to agglutination or clumping of erythrocytes.
    • The Coomb's reagent should be titrated to provide an end-point titer for the reaction. The test may also be performed with individual antisera specific for equine IgG or IgM and may be performed at both 4 and 37°C to assess the thermal reactivity of the erythrocyte -associated immunoglobulins.


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  • The erythrocytes in a blood sample are washed thoroughly in isotonic phosphate-buffered saline (PBS) pH 7.0-7.2, and a 2% suspension of washed RBCs, free of plasma, is established.
  • 25 μl of 2-fold dilutions of an equine-specific Coomb’s reagent ranging from 1:5-1:10240 are prepared across one row of a 96-well round-bottom plate in PBS. Negative control wells containing the same volume of PBS are established in a separate row.  Where the test includes separate antisera for equine IgG and IgM, these are similarly titrated across separate rows of the plate.  Duplicate plates are prepared for incubation at 4 and 37°C in some laboratories.
  • 25 μl of the washed 2% erythrocyte solution are aliquoted into the wells. Positive controls using IgG-sensitized red cells are rarely used in veterinary testing because of the difficulty in obtaining the sensitized control RBCs commercially.
  • One plate is incubated at 4°C for 30-60 min and this is used to identify cold agglutinins (cold reactive antibodies). The other plate is incubated at 37°C for 30-60 min to identify warm agglutinins (warm reactive antibodies). There is some controversy in the literature regarding cold-reactive antibody tests as there may be cold-reactive agglutinins in healthy animals. Comparison of the two sets of results is helpful.
  • All the plates are read by visual assessment of the presence of agglutination. All dilutions that are positive for agglutination are recorded and the titer of each reaction is defined.


  • Generally available in larger commercial and institutional laboratories.
  • Contact the laboratory before sending samples.



  • Less than direct immunofluorescence (DIF) flow cytometry (100% versus 58%) as determined in a population of dogs with IMHA.


  • Better than direct immunofluorescence (DIF) flow cytometry (100% versus 87.5%) as determined in a population of dogs with IMHA.

Predictive value

  • Positive and negative predictive values for equine immune-mediated anemia is 100% and 62% respectively. Low levels of cell-bound antibody may be missed by the Coomb's test, leading to a false negative result.

Technique (intrinsic) limitations

  • The test does not identify the class of antibody found attached to the erythrocyte surface unless specific antisera are used. IgM is often implicated in cold-reacting agglutinins and IgG in warm-reacting agglutinins, but this is not definitive.
  • A negative Coomb's test does not rule out an IMHA. A false negative result may be caused by:
    • Insufficient quantity of antibody or complement on the erythrocytes; improper antiglobulin-to-antibody ratio.
    • Corticosteroid Therapeutics: glucocorticoids treatment for greater than one week in duration prior to the testing; improper or warm temperatures that disperse cold agglutinins.
    • Inadequate washing of the RBCs.
    • Low pH wash solutions.
    • Impaired reactivity of the antiglobulin reagent due to contamination or improper storage.
    • Detachment of the antibody or complement from the RBCs due to the sample ageing.
  • In addition, the prozone effect (high concentrations of bound antibody or complement) may also cause a false negative; this effect can be decreased by complete titration of the Coomb's reagents.
  • False positive results may be seen where there is:
    • Contamination of laboratory apparatus or solutions.
    • Clotted blood samples.
    • Collection of blood from infusion lines where dextrose-containing solutions have been administered.
    • Naturally occurring cold autoantibody that can sensitize the cells with complement.
    • In vitro complement binding during sample storage.
    • Blood transfusions within 21 days of testing.
    • The presence of hypergammaglobulinemia.

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.
  • Caviezel L L et al (2014) Comparison of 4 Direct Coombs’ Test Methods with Polyclonal Antiglobulins in Anemic and Nonanemic Dogs for In-Clinic or Laboratory Use .  J Vet Intern Med 28, 583–591 PubMed.
  • Wardrop K J (2005) The Coombs’ test in veterinary medicine: past, present, future.  Vet Clin Pathol 34, 325–334 PubMed.
  • Wilkerson M J  et al (2000) Isotype-Specific Antibodies in Horses and Dogs with Immune-Mediated Hemolytic Anemia.  J Vet Intern Med 14 (2), 190–196 PubMed.