Page 652 - WSAVA2018
P. 652

 25-28 September, 2018 | Singapore
WSV18-0029
CLINICAL PATHOLOGY
USE AND INTERPRETATION OF COOMBS TEST
M. Day1
1University of Bristol, Bristol Veterinary School, Langford, United Kingdom
USE AND INTERPRETATION OF THE COOMBS TEST Emeritus Professor Michael J. Day
BSc BVMS(Hons) PhD DSc DiplECVP FASM FRCPath FRCVS
Asia Veterinary Diagnostics, Kowloon, Hong Kong
School of Veterinary and Life Sciences, Murdoch University, Western Australia
profmjday@gmail.comIntroduction
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 an- tibodies in cases of suspected immune-mediated thrombocytopenia (IMTP; Evans Syndrome) or im- mune-mediated neutropenia (IMNP).
· As part of the diagnosis of systemic lupus erythe- matosus (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 (4oC) 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 37oC. 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 37oC. 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 37oC. 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 4oC. This pattern is more often are 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
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43RD WORLD SMALL ANIMAL VETERINARY ASSOCIATION CONGRESS AND 9TH FASAVA CONGRESS
































































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