Anti-Nuclear Antibody (ANA) Test

The ANA (anti-nuclear antibody) test is a blood test that looks for antibodies to proteins found in the cell nucleus. The nucleus is essentially the “brain centre” of any cell in the body. Many different types of proteins are found in the nucleus, and they can all be targets for an immune attack. The ANA test is a test to see if there are antibodies in the blood that are attacking any proteins that are found in the nucleus.

The ANA test is a screening test for autoimmune diseases including rheumatic (e.g. Systemic Lupus Erythematosus) and non-rheumatic diseases (e.g. autoimmune hepatitis).

The ANA test is not specific to a single disease. A strongly positive ANA test makes it more likely that a person has an autoimmune disease. However, it does not distinguish between different types of autoimmune diseases. If the anti-ANA test is positive, and there are clinical features of an autoimmune disease, other more specific tests will usually be ordered.

Science Behind this Test

Antibodies

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

Antinuclear antibodies

ANA attacks any proteins that are found in the cell nucleus, the “brain centre” of the cell. Almost every cell has a nucleus. There are several types of proteins in the nucleus that have different functions. All of these proteins are potential targets for an immune attack.

Nuclear proteins are usually hidden from the immune system because they are inside the nucleus. Cells are constantly turning over in our body and during this turnover the nuclear contents may be briefly revealed to the immune system. A normal response for the immune system is to make antibodies against these nuclear contents. 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.” Autoimmune means that the body has failed to recognize its own parts as self, which allows the immune system to mount a response against the body’s own cells and tissues. Systemic Lupus Erythematosus (SLE) is an example of an autoimmune disease. In people with SLE, 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 just indicates that there are antibodies in the blood against proteins in the nucleus.

The ANA test is like using a 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 orange juice from a frozen concentrate. 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. In other words, the body has mounted a substantial immune response against nuclear proteins.

Result 2: the pattern

There are several patterns of anti-nuclear antibodies including:

  • Homogenous
  • Speckled
  • Nucleolar
  • Centromere
  • Rim enhancing

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

A positive ANA test means that a person may have an autoimmune disease, depending on the titre. Almost everyone (99% of people) with lupus has a positive high-titre ANA test result. As a general rule, if the ANA test is negative, it’s not lupus. Other examples of rheumatic diseases that might have a positive ANA test include:

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

However, a positive ANA test does not necessarily mean a person has an autoimmune disease. The body normally makes ANA 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. Therefore, 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
    • 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