Anti-Nuclear Antibody (ANA)

The ANA (anti-nuclear antibody) test is a blood test that looks for antibodies that attack proteins found in the nucleus of cells.

The nucleus is essentially the “command centre” or “brain” of any cell in the body. Many different types of proteins are found in the nucleus that perform many different functions. They can all be targets for an immune attack.

The ANA test is used as a screening test for autoimmune diseases including rheumatic diseases such as Systemic Lupus Erythematosus, and for non-rheumatic diseases such as autoimmune liver diseases, autoimmune thyroid diseases, and autoimmune bowel diseases.

The ANA test is not specific to a single disease. A strongly positive ANA test means that it is more likely that a person has an autoimmune disease. However, positive test results do not tell what type of autoimmune disease it is.

If a patient’s anti-ANA test is positive and they have clinical features of an autoimmune disease, other more specific tests will usually be ordered to help reach a diagnosis.

Science Behind this Test

Antibodies

Anti-nuclear antibodies are a type of autoantibody: they are an antibody produced by the immune system and they target something that the body’s own tissues are made out of.

Antinuclear antibodies

Anti-nuclear antibodies attack proteins that are found in the cell nucleus.

Nuclear proteins are usually hidden from the immune system because they are inside the nucleus, and the nucleus is inside the cell.

Cells are constantly turning over in our body and during this turnover the contents of a cell’s nucleus may be briefly revealed to the immune system. When the immune system sees nuclear proteins, a normal response is to try to make antibodies to the proteins. Fortunately the immune system never gets to see the nuclear proteins for too long so they can’t make many antibodies. So you can see that we can all make antibodies to nuclear proteins.

However, when the immune system can “see” the nuclear contents for longer periods of time, it can mount a significant antibody response. When this happens, lots of antibodies are made and they can attack healthy cells.

Autoimmunity

The ANA test is a test of “autoimmunity.” Autoimmunity is when the body has failed to recognize a part of itself, and the immune system has mounted a response against the body’s own cells and tissues.

Systemic Lupus Erythematosus (SLE) is an example of an autoimmune disease. In people with lupus, the body mounts an immune response to specific proteins found inside the nucleus of their cells.

Why the ANA test is done

The ANA test is a simple screening test to look for antibodies in the blood that are attacking proteins in the nucleus. A limitation of the ANA test is that it does not identify which protein the antibodies are attacking in the nucleus. It only indicates that there are antibodies in the blood against proteins in the nucleus.

The ANA test is like using a radar fish finder. A fish finder indicates if there are any fish below the surface of the water, and gives an idea of how many are there. But it does not say anything about the type of fish or what the fish are doing. The same is true for the ANA test. A positive ANA test confirms that there are antibodies against nuclear proteins but it does not identify which proteins are under attack.

How the ANA test works

The ANA test gives two types of results: 1) the titre, and 2) the pattern.

Result 1: the titre

This part of the ANA test gives an estimate of how many anti-nuclear antibodies are present (i.e. the “titre”).

This is done by taking a sample of the liquid part of the blood called the serum and diluting it with a liquid called a diluent. The process is like making juice from a frozen concentrate where one can is diluted into 4 cans of water (the diluent).

For the ANA test, 1 part of the serum is diluted into 40 parts of diluent (called a “1:40” titre). This diluted sample is tested for the presence of ANA antibodies. If the test is positive, a new sample is made at half the strength (1:80) and tested for the presence of ANA. The test is repeated every time it is positive by diluting the sample to 1:160, 1:320, 1:640, 1:1280, and finally 1:2560.

The lowest dilution is 1:2560. When antibodies are present at the lowest dilution, this indicates that there is a very high number of antibodies in the blood, and that the body has mounted a substantial immune response against nuclear proteins.

Result 2: the pattern

There are several patterns of anti-nuclear antibodies. The ANA staining patterns are loosely associated with underlying autoimmune diseases. The patterns seen are as follows:

• Homogenous: The entire nucleus is stained with ANA. This is the most common pattern and can be seen with any autoimmune disease. Homogenous staining can result from antibodies to DNA and histones.
• Speckled: Fine and coarse speckles of ANA staining are seen throughout the nucleus. This pattern is more commonly associated with antibodies to extractable nuclear antigens. This pattern can be associated with Systemic Lupus Erythematosus, Sjögren’s syndrome, Systemic Sclerosis, Polymyositis, and Rheumatoid Arthritis.
• Nucleolar: The ANA staining is seen around the nucleoles inside the nucleus. This can be seen in Systemic Sclerosis.
• Centromere: The ANA staining is seen along the chromosomes. This pattern can be associated with limited systemic sclerosis, primary biliary cirrhosis, and other autoimmune diseases like Raynaud’s Phenomenon.

Homogenous is the most common and the least concerning pattern of ANA.

ANA test results

A positive ANA test means there are antibodies in the blood against proteins in the cell nucleus. The titre gives an idea of how many anti-nuclear antibodies are in the blood.

The test does not say anything about how tightly the antibodies attach to the nuclear proteins or which specific proteins they are attaching to and attacking.

When the ANA test is positive, other tests are required to identify which protein(s) the antibodies are attacking such as the anti-dsDNA test and the ENA panel.

A positive ANA test means that a person may have an autoimmune disease, depending on the titre.

Almost everyone with lupus (99.9% of cases) has a positive ANA test result. As a general rule, if a person’s ANA test is negative, then they do not have lupus.

Other examples of rheumatic diseases that might have a positive ANA test include:

• Rheumatoid Arthritis
• Sjögren’s Syndrome
• Systemic Sclerosis (Scleroderma)
• Inflammatory Myositis (Polymyositis or Dermatomyositis)
• Mixed Connective Tissue Disease (MCTD)
• Overlap of undifferentiated Connective Tissue Diseases

A positive ANA test does not necessarily mean a person has an autoimmune disease. The body normally makes anti-nuclear antibodies for brief periods of time. This is because cells are constantly turning over in the body and during this turnover the nuclear contents are briefly revealed to the immune system.

The ANA test is very sensitive to picking up anti-nuclear antibodies. In a group of 100 randomly selected people, about 30 would have an ANA positive test at the 1:40 titre, about 10 would be positive at the 1:80 titre, and 3 would be positive at a 1:160 titre.

The ANA test’s results are important to consider because:

• It is a first step in screening for an autoimmune disease
• It may give an estimate of the severity of the disease

Regarding disease severity:

• If the ANA titre is high (e.g. 1:640, 1:1280 or 1:2560), this indicates more severe disease
• If the ANA titre is low (e.g. 1:40, 1:80 or even 1:160), there is often no autoimmune disease
• If the ANA titre is in the middle (e.g. 1:320), the result is less clear and should be interpreted in the clinical context
• If the ANA titre is high, other tests may be ordered to determine what type of nuclear protein is being attacked