Antinuclear antibodies (ANAs), also known as antinuclear factor (ANF), are autoantibodies that bind to the contents of the cell nucleus. In healthy individuals, the immune system produces antibodies against foreign proteins (antigens), but not against human proteins (autoantigens). In some cases, antibodies are produced against human antigens. These are known as autoantibodies.
There are various types of ANAs, such as anti-Ro antibodies, anti-La antibodies, anti-Sm antibodies, anti-nRNP antibodies, anti-Scl-70 antibodies, anti-dsDNA antibodies, anti-histone antibodies, anti-nuclear pore complex antibodies, anti-centromere antibodies, and anti-sp100 antibodies. Each of these antibody subgroups binds to different proteins or protein complexes in the nucleus. They can be found in many disorders, including autoimmune diseases, cancer, and infections, with varying prevalence of antibodies depending on the condition. This allows the use of ANAs in diagnosing certain autoimmune disorders, including systemic lupus erythematosus, Sjögren’s syndrome, scleroderma, mixed connective tissue disease, polymyositis, dermatomyositis, autoimmune hepatitis, and drug-induced lupus.
The ANA test detects autoantibodies present in the serum of individuals. Common tests used to diagnose and quantify ANAs include indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). In immunofluorescence, the level of autoantibodies is reported as a titer. This is the highest serum dilution at which the autoantibodies are still detectable. Positive autoantibody titers at a dilution of 1:160 or greater are usually considered clinically significant. Positive titers less than 1:160 occur in 20% of healthy individuals, especially in older adults. Although positive titers of 1:160 or higher are strongly associated with autoimmune disorders, they are also seen in 5% of healthy individuals. Screening for autoantibodies is useful in diagnosing autoimmune disorders, and monitoring levels helps predict disease progression. If there are no other confirmatory clinical or laboratory data, a positive ANA test is rarely useful.
Immunity and Autoimmunity
The human body has many defense mechanisms against pathogens, one of which is humoral immunity. This defense mechanism produces antibodies (large glycoproteins) in response to an immune stimulus. Many immune system cells are required for this process, including lymphocytes (T cells and B cells) and antigen-presenting cells. These cells coordinate an immune response by recognizing foreign proteins (antigens) and producing antibodies that bind to these antigens. In normal physiology, lymphocytes that recognize human proteins (autoantigens) undergo either programmed cell death (apoptosis) or become nonfunctional. This self-tolerance means that lymphocytes should not stimulate an immune response against human cellular antigens. However, sometimes this process malfunctions, and antibodies are produced against human antigens, which may lead to autoimmune disease.
ANA Subgroups
ANAs are found in many disorders as well as in some healthy individuals. These disorders include systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjögren’s syndrome, scleroderma, polymyositis, dermatomyositis, primary biliary cirrhosis, drug-induced lupus, autoimmune hepatitis, multiple sclerosis, discoid lupus, thyroid disease, Juliette syndrome, anti-phospholipid syndrome, psoriatic arthritis, juvenile dermatomyositis, idiopathic thrombocytopenic purpura, infections, and cancer. These antibodies can be categorized based on their characteristics, and each subset has a different propensity for specific disorders.
Extractable Nuclear Antigens
Extractable nuclear antigens (ENA) are a group of autoantigens that were initially identified as antibody targets in individuals with autoimmune disorders. They are called ENA because they can be extracted from the cell nucleus using salt. ENAs consist of ribonucleoproteins and non-histone proteins, named after the donor of the initial serum sample (Sm, Ro, La, Jo) or named according to the disease environment in which the antibodies were found (SS-A, SS-B, Scl-70).
ANA Test
The presence of ANAs in the blood can be confirmed through screening tests. Although many tests exist for detecting ANAs, the most commonly used tests for screening are indirect immunofluorescence and ELISA. Once ANAs are diagnosed, various types of them are specified.
Indirect Immunofluorescence
Indirect immunofluorescence is one of the most commonly used tests for ANAs. Typically, HEp-2 cells are used as a substrate to detect human serum antibodies. Microscopic slides are coated with HEp-2 cells, and serum is incubated with the cells. If the specified and targeted antibodies are present, they bind to the antigens on the cells. In the case of ANAs, the antibodies bind to the nucleus. These can be visualized by adding a fluorescent-labeled anti-human antibody (usually FITC or rhodamine B) that binds to the antibodies. This molecule fluoresces when exposed to a specific wavelength of light, which can be observed under a microscope. Depending on the antibody present in human serum and the location of the antigen in the cell, distinct patterns of fluorescence are seen on the HEp-2 cells. The level of antibodies is analyzed by performing dilutions on the serum. If fluorescence is observed at a titer of 1:40/1:80, the ANA test is considered positive. Higher titers are clinically more significant because low positive cases (≤1:160) are found in 20% of healthy individuals, especially older adults. Only about 5% of healthy individuals have an ANA titer of 1:160 or higher.
Until about 1975, when HEp-2 cells were introduced, animal tissue was used as the standard substrate for immunofluorescence. HEp-2 cells are now one of the most common substrates for detecting ANAs by immunofluorescence. They are preferred due to their larger size and high mitotic rate (cell division) compared to the previously used animal tissues. This allows for the identification of specific antigen-antibody interactions, such as centromere antibodies. They also enable the identification of anti-Ro antibodies, as acetone is used to fix the cells (other fixatives can wash out antigens).
ELISA
The enzyme-linked immunosorbent assay (ELISA) uses microtiter plates coated with antigens to detect ANAs. Each well of a microtiter plate is sequentially coated with a single or multiple antigens to identify specific antibodies or screen for ANAs. The antigens may be from cellular extracts or recombinant sources. Serum is incubated in the wells and then washed. If antibodies that bind to the antigen are present, they remain after washing. A secondary anti-human antibody conjugated to an enzyme such as horseradish peroxidase is added. The enzymatic reaction produces a change in the color of the solution, which corresponds to the amount of antibody bound to the antigen. There are significant differences in the detection of ANAs by immunofluorescence and various ELISA kits, with only marginal agreement between them. A physician should be familiar with these differences to accurately assess the results of different assays.
Sensitivity
Some ANAs appear in several types of diseases, resulting in lower test specificity. For example, IgM rheumatoid factor (IgM-RF) has been shown to cross-react with ANAs, leading to false-positive immunofluorescence results. Positive ANAs and anti-DNA antibodies have also been reported in patients with autoimmune thyroid disease. ANAs can yield a positive test result in 45% of individuals with autoimmune thyroid conditions or rheumatoid arthritis and up to 15% of individuals with HIV or hepatitis C. According to the Lupus Foundation of America, “approximately 5% of the general population will be ANA positive. However, at least 95% of individuals who are ANA positive do not have lupus. A positive ANA test can sometimes run in families, even if family members show no evidence of lupus.” On the other hand, they say that although 95% of patients who actually have lupus test ANA positive, “only a small percentage are ANA negative, and many patients with other antibodies (such as anti-phospholipid, anti-Ro, anti-SSA antibodies) may have their ANAs turn from positive to negative due to steroids, cytotoxic drugs, or uremia (kidney failure).”
History
The LE cell was discovered in 1948 by Hargraves and colleagues in the bone marrow. In 1957, Halbur et al. were the first to show ANAs using indirect immunofluorescence. This was the first indication that processes affecting the cell nucleus were responsible for SLE. In 1959, it was discovered that the serum of individuals with SLE contained antibodies that precipitate with saline extracts of nuclei, known as extractable nuclear antigens (ENA). This led to the identification of ENA antigens and their corresponding antibodies. Thus, anti-Sm and anti-RNP antibodies were identified in 1966 and 1971, respectively. In the 1970s, anti-Ro/anti-SS-A and anti-La/anti-SS-B antibodies were discovered. Scl-70 antibodies were recognized as a specific antibody for scleroderma in 1979, although the antigen (topoisomerase-I) was not identified until 1986. The Jo-1 antigen and antibody were identified in 1980.
