This lecture will use a case study to demonstrate the use and interpretation of the Coombs test in canine medicine. The Coombs test might be requested in the following clinical situations:

• To confirm the presence of red cell-bound IgG, IgM, or complement C3 in suspected cases of primary immune-mediated haemolytic anaemia (IMHA).
• To confirm an immune-mediated component in cases of secondary IMHA (e.g., infection, neoplasia, drug or vaccine exposure).
• To determine the presence of red cell-bound antibodies in cases of suspected immune-mediated thrombocytopenia (IMTP; Evans Syndrome) or immune-mediated neutropenia (IMNP).
• As part of the diagnosis of systemic lupus erythematosus (SLE; a very rare disease), when anaemia is one clinical problem.
• To diagnose cold agglutinin disease.

The Coombs Test

The Coombs test is used to demonstrate the presence of antibody and/or complement proteins on the surface of red blood cells (RBCs) in order to confirm a diagnosis of immune-mediated haemolytic anaemia (IMHA). In veterinary medicine, a direct Coombs test is performed, using RBCs from the patient. Detecting free serum antibodies specific for RBCs (the indirect Coombs test) is not performed diagnostically.

The sample required for a Coombs test is 2–5 ml EDTA anticoagulated blood (volume determined by the degree of anaemia). There are no specific handling requirements and the sample can be sent as for standard haematological examination. The attachment of antibody to RBCs is relatively robust and the test will still be positive after shipment. The test will also be unaffected if an animal has just started immunosuppressive therapy.

The first stage of the test, which may also be simply performed in practice, is the ‘in saline agglutination test.’ The tube of EDTA blood should be placed in a refrigerator (4°C) for 30 minutes and then evaluated for agglutination of RBCs. If there is visible evidence of agglutination, a drop of blood is placed on a microscope slide and diluted with a drop of saline. The slide may be gently rocked to mix the blood and saline. If the aggregated RBCs disperse, they are not true agglutinates (and are likely rouleaux formed in hyperproteinaemic plasma), but if they persist, then this is evidence of autoagglutination. This reaction may not be seen at room temperature or 37°C. The observation of autoagglutination is highly indicative of IMHA, but occurs perhaps only for one in every 10 dogs with the disease. The presence of autoagglutination does not necessarily preclude performing the Coombs test.

The Coombs test is a simple agglutination test. RBCs from the patient are ‘washed’ in phosphate-buffered saline (PBS) to remove the plasma and buffy coat, and the cells are then resuspended in PBS. Washed RBCs are then mixed with an antiserum (or a panel of different antisera) with specificity for the immunoreactants that might be expected to be found on the surface of RBCs in IMHA (i.e., IgG, IgM, or complement C3). The antiserum will cross-link molecules of immunoglobulin or complement on the surface of the RBCs providing a visual read-out as agglutination.

Different diagnostic laboratories perform the Coombs test in different ways. Some use only one antiserum (the polyvalent canine Coombs reagent) to detect any immunoreactants present. The most thorough Coombs test uses polyvalent reagent in addition to separate specific antisera for IgG, IgM, and complement C3, and fully titrates these antisera, while duplicating the test for incubation at both 4 and 37°C. Such testing provides much more complete information about the nature of the immunological reaction occurring.

In general, there are two broad patterns of Coombs test reactivity and these correlate well with the clinical presentation, severity of disease, and prognosis. The most common pattern indicates the presence of a ‘warm- reactive IgG antibody’ where the test is positive for IgG alone, IgG + IgM (with or without complement) with similar titres at 4 and 37°C. This pattern is generally seen with dogs that have chronic compensated disease caused by extravascular haemolysis that carries a better prognosis. The less common pattern is that of ‘cold-reactive IgM antibody’ where the test is dominated by IgM antibody (with or without complement) with higher titre at 4°C. This pattern is more often associated with acute onset disease, autoagglutination, intravascular haemolysis, and a more guarded prognosis. Sometimes a mixed pattern of Coombs reactivity occurs in a single dog.

The Coombs test does not distinguish between primary and secondary IMHA, but unusual patterns, low-titre reactions, and cold-reactive IgM are more likely to be associated with secondary disease.

The chances of a false-negative Coombs test are minimized by performing the complete test as described above. However, it is possible for rare cases to have insufficient RBC-associated immunoreactants for detection or for circulating RBCs to be unaffected where the immune attack is directed against erythroid precursors in the bone marrow (non-regenerative IMHA or pure red cell aplasia).

In some laboratories, detection of RBC-bound immunoreactants is performed using fluorochrome-labelled antisera and the RBCs are passed through the laser beam of a flow cytometer. Flow cytometry is reported to be equally or more sensitive than the Coombs test, but the Coombs test is considered to be more specific than flow cytometry.

Some in-house rapid diagnostic tests have also been developed that provide a yes/no answer (i.e., is the dog Coombs positive?) within 30 minutes. Currently, in Europe the most widely available of these tests is that marketed by Alvedia (LabTest DAT). Not all of these tests have been validated against the gold standard full test (as described above), but those that have often show good correlation.

Further Reading

1.  Caviezel LL, Raj K, Giger U. 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. 2014;28:583–591.

2.  Day MJ. Arthropod-borne infectious diseases of the dog and cat, 2nd ed. London, UK: Taylor and Francis; 2016.

3.  Day MJ, Kohn B, ed. Manual of Canine and Feline Haematology and Transfusion Medicine, 2nd ed. Gloucester, UK: BSAVA; 2012.

4.  Day MJ. Clinical Immunology of the Dog and Cat, 2nd ed. London, UK: Manson Publishing Ltd.; 2012.

5.  Day MJ, Schultz RD. Veterinary Immunology: Principles and Practice, 2nd ed. London, UK: Manson Publishing Ltd.; 2014.

6.  Harkin KR, Hicks JA, Wilkerson MJ. Erythrocyte-bound immunoglobulin isotypes in dogs with immune-mediated hemolytic anemia: 54 cases (2001–2010). J Am Vet Med Assoc. 2012;241:227.

7.  Morley P, Mathes M, Guth A, et al. Anti-erythrocyte antibodies and disease associations in anemic and non-anemic dogs. J Vet Intern Med. 2008;22:886–892.

8.  Piek CJ, Teske E, van Leeuwen MW, et al. Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia. Acta Vet Scand. 2012;54:10.

9.  Warman SM, Murray JK, Ridyard A, et al. Pattern of Coombs test reactivity has diagnostic significance in dogs with immune-mediated haemolytic anaemia. J Small Anim Pract. 2008;49:525–530.