What is ANF?
The test called ANF (antinuclear factor) or ANA (antinuclear antibody) is a test commonly ordered for patients who are suspected of having an autoimmune disease.
Under normal conditions, the immune system responds to germs invading our bodies by producing a large number of antibodies to fight them off. When a person has an autoimmune disease, the body acts inappropriately and produces antibodies against the body’s own cells, tissues, and proteins as if they were the aggressor. ANF is one of these autoantibodies that are produced in autoimmune diseases.
ANF is actually not a single antibody, but rather a group of autoantibodies that were discovered in the 1940s in patients with systemic lupus erythematosus. As the name already suggests, ANF are antibodies against the nuclei of our cells. There are several types of ANF, each one directed against a specific cell structure and associated with a different type of autoimmune disease.
It is important to note that 10% to 15% of the healthy population may have positive ANF at low values without this indicating any health problem. We do not know the reason for this finding, but the simple presence of a positive ANF is not sufficient for the diagnosis of any disease.
What is an autoimmune disease?
Autoimmune diseases are those that occur when our immune system mistakenly fails to recognize some of the structures present in our body and starts treating them as if they were harmful invading germs. The result of this confusion is the production of the so-called auto-antibodies, that is, antibodies against ourselves.
Autoantibodies, unlike normal antibodies, do not fight bacteria, viruses, parasites or fungi. They attack our cells, destroying them. Autoimmune diseases can affect the cells of the blood, skin, joints, kidneys, lungs, nervous system, etc. Making an analogy, we can say that an autoimmune disease is a kind of mutiny carried out by our own security forces.
Some examples of autoimmune diseases include:
- Rheumatoid arthritis.
- Multiple sclerosis.
- Hashimoto’s thyroiditis.
- Wegener’s granulomatosis.
- Guillain Barré syndrome.
- Myasthenia gravis.
How is the ANF test performed?
To understand the results of the ANF is necessary to know how the test is done. The subject is quite complex, but I will try to explain as simply as possible.
The ANF test is done on blood samples from the patient suspected of having an autoimmune disease. In the laboratory, we are able to identify all of the antibodies circulating in the blood by adding a fluorescent dye to the blood. After the antibodies are labeled, we mix the blood in a container with a culture of human cells (called Hep2 cells).
The result is what you see in the picture above. If there are antibodies against structures of the human cells, these will attach themselves to the cells, making them fluoresce. If the autoantibody is against the nucleus of the cells, the image in the microscope will be of several fluorescing nuclei. If the autoantibody is against the cytoplasm of the cells, several cytoplasms will glow, and so on. If there is no autoantibody, no part of the cells will fluoresce, characterizing a non-reactive ANF.
Results are repeated after several dilutions of the blood until the fluorescence disappears. Positive results are those that remain glowing even after 40 dilutions (1/40 or 1:40 result). Therefore, a 1/40 reactive ANF means that the autoantibody has been identified even after diluting the blood 40 times.
As I explained before, up to 10% of the population is ANF positive, usually in dilutions smaller than 1/80. For this reason, we only consider values starting at 1/160 as relevant. Dilutions are usually done in the following order: (1/40), (1/80), (1/160), (1/320), (1/640), (1/1280)… Values greater than or equal to 1/320 are very relevant and indicate autoimmune disease in more than 97% of cases.
From now on, you can already have an idea of the results of ANF. Let’s cite some examples:
ANF (Hep2): Reagent.
Pattern: nuclear fine dotted.
2) ANF (Hep2): Reagent.
Pattern: cytoplasmic dotted reticulated.
The two examples above are of a reactive ANF. The first at low titers, with the nucleus being stained with fine fluorescent dots. This first example does not necessarily indicate the presence of autoimmune disease. The second example shows a positive ANF at high titers, with antibodies against the cytoplasm of the cell forming a cross-linked image.
Types of reactive ANF
There are more than 20 different Immunofluorescence standards. Each one describes the way human cells were stained by fluorescent antibodies. Some patterns are typical of diseases, such as lupus, scleroderma, rheumatoid arthritis and Sjögren’s syndrome. Others are nonspecific and may be present in normal people.
The most common ANF patterns and their likely pathologies, are:
- Centromeric punctate nuclear: Scleroderma or primary biliary cirrhosis.
- Nuclear homogeneous: Lupus, Rheumatoid Arthritis, Juvenile Idiopathic Arthritis, Felty Syndrome or Primary Biliary Cirrhosis.
- Nuclear continuous membrane type: Lupus or autoimmune hepatitis.
- Fine dotted nuclear: Primary Sjögren’s Syndrome, Systemic Lupus Erythematosus or Lupus.
- Fine Dense Punctate Nuclear: Nonspecific, may be present in several autoimmune diseases and also in Interstitial Cystitis, Atopic Dermatitis, Psoriasis or Asthma.
- Thick dotted nuclear: Mixed Connective Tissue Disease, Systemic Lupus Erythematosus, Systemic Sclerosis or Rheumatoid Arthritis.
- Dotted nucleolar: Systemic Sclerosis.
- Reticular dotted cytoplasmic: Primary Biliary Cirrhosis or Systemic Sclerosis.
- Fine dotted cytoplasmic: Polymyositis or Dermatomyositis.
You need to know how to interpret antinuclear factor results. The same pattern can mean several different autoimmune diseases or nothing at all.
The ANF needs to be evaluated along with the patient’s clinical picture. It is important to remember that the patient is a whole and not just a result of a chemical reaction printed on a piece of paper. The associations described above are possibilities and not fait accompli. I have received in the comments dozens of requests for interpretation of ANF based solely on its results. Antinuclear factor alone is not diagnostic of anything. This is a test that cannot be evaluated from a distance. It is only one piece of the puzzle.
The diseases most associated with positive antinuclear factor are systemic autoimmune diseases, such as Lupus and Scleroderma (systemic sclerosis). ANF can also be positive in autoimmune diseases restricted to a few organs, such as Hashimoto’s thyroiditis and autoimmune hepatitis.
We call sensitivity the ability of a test to test positive when the patient has the disease. For example, when we say that the sensitivity of ANF for lupus is 95%, that means that for every 100 patients with lupus who are tested, 95 will test positive and only 5 will test false negative. So, if your doctor suspects that you may have lupus but the ANF result is negative, it means that he has to start thinking about another diagnosis because the chance of you having lupus is less than 5%.
The sensitivity of ANF in major autoimmune diseases is as follows:
- Lupus: 95 to 100%.
- Scleroderma: 60 to 80%.
- Mixed connective tissue disease: 100%.
- Polymyositis and dermatomyositis: 61%.
- Rheumatoid arthritis: 52%.
- Sjögren’s syndrome: 40 to 70%.
- Drug-induced lupus: 100%.
- Lupus discoid: 15%.
- Hashimoto’s thyroiditis: 46%.
- Autoimmune hepatitis: 100%.
- Primary autoimmune cholangitis: 100%.
ANF false positive
Like any laboratory test, ANF can also have false positives. 10 to 15% of the healthy population may have false positive ANF, mainly women and the elderly. When we evaluate only the population older than 65 years, the rate of false positive ANF reaches 30%.
Some diseases or medications can also cause a reactive ANF without signifying the presence of an autoimmune disease. Among the diseases that can cause a positive ANF, the most common are HIV, mononucleosis, lymphoma, and tuberculosis. Among the drugs, the most common are hydralazine, isoniazid and procainamide.
What to do in case of a positive ANF?
Once you have a positive ANF associated with a clinical picture that suggests autoimmune disease, you should order specific autoantibodies to try to define exactly which autoimmune disease you are dealing with.
The ANF suggests the presence of an autoantibody, but does not define exactly which one. For example, a ANF suggestive of lupus needs to be complemented with an anti- native DNA antibody, which is the typical lupus autoantibody; in suspected Sjögren’s, anti SS-A/Ro and anti SS-B/La antibodies are requested; in scleroderma, anti-centromere antibody is dosed, and so on.
Often, the patient is ANF positive but has negative specific autoantibodies. In these cases there are usually two possibilities: either the ANF is false positive or the patient may have an autoimmune disease that is not yet active. The patient has the autoantibodies, but they are not yet attacking the body. There are patients with lupus who have a reactive ANF for years before the disease manifests itself clinically. There are others, however, who have positive ANF for the rest of their lives and never develop any health problems.
In conclusion, antinuclear factor is an initial test in the investigation of autoimmune diseases. ANF alone does not close any diagnosis. When the patient has symptoms typical of autoimmune disease, it helps to guide the diagnosis.
On the other hand, when the patient is asymptomatic, a positive result will do more harm than good. Therefore, it does not make sense to order the ANF test unless there is a strong suspicion of autoimmune disease.
- When to order an antinuclear antibody test – British Medical Journal.
- Know Your Labs – The Rheumatologist.
- Lupus Blood Tests – Johns Hopkins Rheumatology.
- Antinuclear Antibodies (ANA) – American College of Rheumatology.
- Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015 – Frontiers in immunology.