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Note: Because of the technical nature of this topic, you may wish to consult with your physician if you have questions after reading this information

Our immune system protects us from infection by producing antibodies to fight off or neutralize harmful bacteria, viruses, and other microbes. Antibodies bind to components (usually proteins) of the microbes called antigens. When these antibodies malfunction and attach to antigens produced by the body’s own cells, the immune system may attack one’s own body, leading to autoimmune (self-immune) diseases such as rheumatoid arthritis, systemic lupus erythematosus, scleroderma, polymyositis, Sjögren’s syndrome and vasculitis. Standard FANA blood tests are used to screen for most of these specific systemic autoimmune disorders, though not for rheumatoid arthritis.

By itself a positive FANA test does not indicate the need for therapy.

While a “false positive” FANA test typically signals the presence of antinuclear antibodies in a healthy individual, these antibodies are usually of no clinical significance and are best ignored.

Although a positive FANA test is not uncommon in RA, there is little clinical significance unless an autoantibody specific to a particular disease is found.

The Fluorescent Antinuclear Antibody (FANA) (sometimes abbreviated to ANA) test is a blood test used to search for the presence of abnormal antibodies. These abnormal antibodies, called autoantibodies, bind to components of the person’s own cells calls “antigens,” causing the immune system to attack the body in an effort to combat disease. To measure results of the FANA test, human tissue culture cells (HEp-2 cell line) are grown in culture and allowed to stick to a microscope slide. Next the slides are treated with methyl alcohol to “fix” the cells and make them permeable before being incubated with the patient’s blood. The slide is then washed to remove unbound antibodies, followed by incubation with fluorescent antibodies that detect the binding of human antibodies to the cells. Finally the slide is viewed using a fluorescence microscope, and staining intensity and the pattern of binding are scored at various dilutions.   How the FANA test is read Typically FANA test results are reported in titers (how much the patient’s blood was diluted) and patterns, e.g., homogeneous, speckled, nucleolar, centromere, cytoplasmic, etc.

The titer reading is determined by diluting the liquid portion of a person’s blood or serum in saline (see Figure above). In the example, 1 part serum is mixed with 40 parts saline to create a 1:40 dilution. Next, more saline is mixed with the 1:40 dilution (1 part diluted serum plus 1 part saline) creating a new 1:80 sample (just one tube difference even though the doubled number reference sounds much higher). The dilution then is taken through a series of additional steps, creating tubes of 1:160, 1:320, and 1:640 dilutions, respectively.

As indicated above, the diluted serum samples are incubated with HEp-2 cells and antibody binding is detected with fluorescent antibodies. If a fluorescent signal (green in the Figure) is seen in the nucleus, then the diluted serum sample contains antinuclear antibodies. At some point in the process as more dilution results in a lower concentration of antinuclear antibodies, the fluorescent signal will no longer be visible. In the example, the fluorescent signal seen in 1:40 serum is still visible at 1:80, 1:160, and 1:320, but not at 1:640.

The “titer” reported by the laboratory is the last dilution giving a positive signal— in this example 1:320. Thus, the higher the amount of dilution at which antinuclear antibodies can be found, the more antinuclear antibodies are in the person’s blood— e.g. a titer of 1: 320 means a higher concentration of antinuclear antibodies than a titer of 1: 160. Remember, a titer of 1:320 is only 1 tube different than a titer of 1:160, so this is generally not a significant difference in antinuclear antibody level. A 2-tube difference (e.g. 1:160 vs. 1:640), however, usually is a significant difference.

The patterns correspond to different antigens that have bound to autoantibodies in the patient’s blood. Homogeneous, speckled, nucleolar and centromere patterns refer to staining of different parts of the cell nucleus, whereas cytoplasmic staining refers to staining of antigens in the cytoplasm. Each pattern is significant. For instance, a nucleolar pattern suggests a diagnosis of diffuse cutaneous systemic sclerosis (scleroderma), whereas a centromere pattern is associated with the limited cutaneous form of systemic sclerosis (scleroderma), in particular the CREST syndrome (an acronym standing for Calcinosis, Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasias). Homogeneous and speckled nuclear and cytoplasmic patterns are less disease-specific.

How the FANA test is used

Often this type of testing is used to help screen for certain systemic autoimmune diseases such as SLE, polymyositis, scleroderma and Sjögren’s syndrome.

The FANA’s sensitivity and simplicity makes it extremely popular to screen for SLE in particular. Because approximately 90-95% of individuals with SLE will have a positive test, a negative FANA test can be helpful in excluding that diagnosis. In contrast to the high sensitivity, the specificity of a positive ANA for SLE is relatively low.

Thus, only about 11-13% of persons with a positive FANA test have SLE. In one study of ANA-positive individuals, 19% had SLE, 11% drug-induced lupus, 22% other systemic autoimmune diseases (e.g., Sjögren’s syndrome, polymyositis, scleroderma), 10% autoimmune thyroiditis, 6% other organ specific autoimmune diseases , 8% infections, 3% neoplasms and 24% other conditions or “idiopathic” autoantibodies.

What causes a false positive FANA test

As discussed above, most patients with a “false positive” FANA test actually do have antinuclear antibodies. However, not all antinuclear antibodies are clinically significant.

Further immunological testing may reveal disease-specific autoantibodies such as those binding to the antigens double-stranded DNA or Sm (specific for SLE), topoisomerase I (Scl-70) (specific for scleroderma) and Jo-1 (specific for polymyositis). Antinuclear antibodies that are not clinically significant generally react with different cellular components (antigens) than those recognized by the disease-specific autoantibodies. They may result from viral infections (e.g., parvovirus B19, Epstein-Barr virus, cytomegalovirus), bacterial or parasitic infections (e.g., bacterial endocarditis, leprosy, malaria), tumors (e.g., atrial myxoma, a tumor of the heart), drugs (e.g., minocycline, nitrofurantoin, methyldopa, hydralazine), or cytokines (proteins that send signals to immune cells) such as interferon α, tumor necrosis factor α, and interleukin (IL) 6. The antinuclear antibodies developing in healthy elderly individuals may be caused by dysregulated production of cytokines. A positive FANA also is common in patients with autoimmune thyroid disease (Hashimoto thyroiditis).

Healthy individuals may well register a “false positive” in the FANA test, but this result is usually of no clinical significance.

Additional immunological testing often will be helpful following a positive FANA test to help diagnose autoimmune diseases more specifically.

Although the FANA test is a standard test, alternatives such as the enzyme-based immunoassay (EIA or ELISA) have become available and are more amenable to automation. However, they are generally less reliable.

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