Canis ISSN: 2398-2942

Direct Coombs' test

Synonym(s): Direct antiglobulin test

Contributor(s): Vetstream Ltd


  • Theoretically detects presence of antibodies directed against erythrocytes, eg in immune-mediated hemolytic anemia Anemia: immune mediated hemolytic.
  • Best interpreted as evidence that there is detectable immunoglobulin and/or complement bound or adsorbed to erythrocyte membranes.
  • May also be carried out at 4°C to detect cold-reacting antibodies: controversial.


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Adsorption of non-specific agglutinins

  • Coombs' antiserum consists of a polyvalent rabbit antiserum containing antibodies directed against IgG, IgM and complement C3.
  • Incubate this with patient's RBCs → agglutination then RBCs are coated with IgG, IgM and/or complement.
  • Prior to test, inactivate the rabbit (Coombs') antiserum against washed normal canine erythrocytes for 1 hour at 37°C then overnight at 4°C.

Direct Coombs' test

  • Prepare a 1% suspension of washed RBCs:
    • Pipette doubling dilutions of Coombs' antiserum into microtitre plate wells, eg dilutions from 1:2 up to 1:2048.
    • Add 50 microliters of RBC suspension and allow to settle.
    • Run a control in parallel containing only RBCs.
    • Incubate at 37°C for 30 minutes.
  • RBCs form a red dot at bottom of well → negative result.
  • Small, red suspended specks → agglutination → positive result.
  • Record the maximum dilution at which agglutination is seen.
  • Interpret positive titers <1:16 dilution cautiously, especially without evidence of overt hemolysis.
  • Titers of 1:32 and above are definitely significant.



  • 60-70% of auto-immune hemolytic anemia cases are Coombs-positive.


  • Reasonable, but several reasons for false positives.

Technique (intrinsic) limitations

  • Definitive diagnosis of anti-erythrocyte antibodies.
  • Definitive diagnosis of auto-immune hemolytic anemia.
  • Needs more evaluation in dogs.

Result Data

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Further Reading


Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Day M J (1996) Serial monitoring of clinical, hematological and immunological parameters in canine auto-immune hemolytic anemia. JSAP 37(11), 523-534.
  • Jones D R et al (1987) An enzyme-linked antiglobulin test for the detection of erythrocyte-bounc antibodies in canine auto-immune hemolytic anemia. Vet immunol Immunopathol 16(1-2), 11-21.