Equis ISSN 2398-2977

Direct Coombs test

Synonym(s): Direct antiglobulin test, DAT

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

Overview

  • 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.

Sampling

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Tests

Methodologies

  • 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.

Availability

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

Validity

Sensitivity

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

Specificity

  • 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

Publications

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.


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