Mix the drop of saline with one drop of the patient's anticoagulated blood and observe for the presence of agglutination under the microscope. Place one drop of 0.9% saline on a microscope slide. To perform a saline agglutination test: 1. The saline agglutination test is simple to perform in house and aids in the diagnosis of immune-mediated hemolytic anemia (IMHA). Hemagglutination inhibition (HI) is used to determine antibody titers to canine parvovirus and canine influenza virus, and it evaluates the ability of serum to inhibit erythrocyte agglutination by these viruses. Examples of agglutination tests that are used to diagnose infectious diseases of dogs and cats include the microscopic agglutination test (MAT) for serologic diagnosis of leptospirosis (agglutination of live leptospires) and the cryptococcal antigen latex agglutination test (agglutination of antibody-coated latex beads). This is called the prozone effect and can result in false-negative test results if serum is not adequately diluted. If excess antibody is present, particles may become so coated with antibody that agglutination is actually inhibited. IgM causes agglutination more effectively than IgG.
Serial dilutions of serum are tested for their ability to cause or inhibit agglutination, and the highest dilution that causes or inhibits agglutination is reported as the antibody or antigen titer. They rely on the bivalent nature of antibodies, which can cross-link particulate antigens. Rankin, in Canine and Feline Infectious Diseases, 2014 Agglutination TestingĪgglutination tests detect antibody or antigen and involve agglutination of bacteria, red cells, or antigen- or antibody-coated latex particles. To achieve maximum reliability and standardization, laboratories are also encouraged to participate in quality assurance programmes, such as the International Leptospirosis MAT proficiency test scheme, organised by the International Leptospirosis Society (ILS). A large panel of leptospire strains must be routinely maintained and updated for new, recently isolated serovars. The complexity of this test limits its use to reference laboratories. The MAT is considered the gold standard method for immune phase diagnostics by the World Health Organization. These reactions are common however, they become less pronounced with the maturation of the immune response, which increases the test specificity. However, cross-reaction of the patients’ antibodies with different serovars may produce paradoxical reactions.
The agglutination reaction is visualised by dark field microscopy, and the end-point is considered as the serum dilution that promotes 50% of agglutination. The MAT titre of a patient is determined by the incubation of different serum dilutions with a panel of leptospires. Angela Silva Barbosa, in Molecular Medical Microbiology (Second Edition), 2015 MAT