The antinuclear antibody test is an assay that detects the presence of antibodies that bind to nuclear antigens.
The antinuclear antibody test is an assay that detects the presence of antibodies that bind to nuclear antigens. These include DNA, RNA, histones, and other nuclear proteins. The standard ANA test was developed using slices of rat liver as substrate in an indirect immunofluorescence assay using patient serum as the primary antibody. The technique evolved to use a cell line (Hep2 cells) in place of a tissue section. Normally serum is diluted 1:20 and two fold thereafter. Incubation with cells is followed by washing and addition of a species specific anti-globulin conjugated to fluorescein. The presence of nuclear fluorescence is compared with positive and negatively stained control wells and a titer is determined after sequential observation of the fluorescence apparent with each serum dilution. A positive ANA test is shown in Figure 1.
Figure 1. The presence of antinuclear antibodies (ANA) is associated with autoimmune disease, specifically systemic lupus erythematosus, when present at high titers (>100). At lower titers a positive ANA may be insignificant. Associations with tick borne diseases, old age, and recent major trauma have been documented for low titer ANA.
The ANA test shown above illustrates a homogeneous pattern of staining. Other patterns seen include speckled, nucleolar and rim. In human autoimmune disease different patterns of fluorescence correlate with specific autoimmune diseases. In veterinary autoimmune disease the correlations are less specific. In a recent publication Hansson-Hamlin et al describe subgroups of autoimmune disease that are characterized by different patterns of immunofluorescense on the ANA test. In 56 dogs with ANA titers greater than 100 they found that 27% of the dogs showed a chromosomal-positive, homogeneous ANA staining pattern and clinical signs related to multiple organ systems, including anemia. Seventy five percent of the dogs had a speckled ANA staining pattern and these showed clinical signs that were mainly related to the musculoskeletal system. In another study Smee et al evaluated ANA titers in dogs with systemic lupus erythematosus and disorders with related clinical signs. In their study of 120 dogs the most common clinical signs were arthralgia, myalgia, and stiffness (34.2%). Twenty five percent of the dogs had thrombocytopenia. The serum ANA titer was < 160 in 74.2%, 160 in 11.7%, 320 in 4.2%, and > or = 640 in 10%. From this study the authors concluded that results of the ANA assay are likely to be positive and compatible with immune-mediated disease if at least 2 major clinical signs were evident. These results are in line with the criteria used by the American Rheumatology Association to classify diagnosis of SLE in human patients.
Histones are a nuclear component that constitutes an important antigen in positive ANA tests. In one study Ginel et al performed assays for anti-histone antibodies on dogs with leishmaniasis. They found an association between serum anti-histone antibodies and glomerulonephritis in a group of 43 dogs examined. Statistical analysis showed that dogs with high anti-histone antibody titers were more likely to have glomerularnephritis than other dogs with leishmaniasis.
1. Ginel PJ, Camacho S, Lucena R. Anti-histone antibodies in dogs with leishmaniasis and glomerulonephritis. Res Vet Sci. 2008 Dec;85(3):510-4.
2. Hansson-Hamlin H, Lilliehöök I, Trowald-Wigh G. Subgroups of canine antinuclear antibodies in relation to laboratory and clinical findings in immune-mediated disease. Vet Clin Pathol. 2006 Dec;35(4):397-404.
3. Smee NM, Harkin KR, Wilkerson MJ. Measurement of serum antinuclear antibody titer in dogs with and without systemic lupus erythematosus: 120 cases (1997-2005). J Am Vet Med Assoc. 2007 Apr 15;230(8):1180-3.