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Antinuclear Antibodies Direct

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Antinuclear Antibodies Direct


Overview:
Antinuclear antibodies (ANA) are (auto)antibodies that are reactive with antigens in the nucleoplasm. These antibodies probably occur in the circulation of all human beings, but the employed test is only considered ‘positive’ if they occur at titres elevated significantly above the normal serum level. ANA were first demonstrated in 1957 by Holborow et al., using indirect immunofluorescence.
We normally have antibodies in our blood that repel invaders into our body, such as virus and bacteria microbes. Antinuclear antibodies (ANAs) are unusual antibodies, detectable in the blood, that have the capability of binding to certain structures within the nucleus of the cells. The nucleus is the innermost core within the body's cells and contains the DNA, the primary genetic material. ANAs are found in patients whose immune system may be predisposed to cause inflammation against their own body tissues. Antibodies that are directed against one's own tissues are referred to as auto-antibodies. The propensity for the immune system to work against its own body is referred to as autoimmunity. ANAs indicate the possible presence of autoimmunity and provide, therefore, an indication for doctors to consider the possibility of autoimmune illness.

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Antinuclear Antibodies Direct

Overview:
Antinuclear antibodies (ANA) are (auto)antibodies that are reactive with antigens in the nucleoplasm. These antibodies probably occur in the circulation of all human beings, but the employed test is only considered ‘positive’ if they occur at titres elevated significantly above the normal serum level. ANA were first demonstrated in 1957 by Holborow et al., using indirect immunofluorescence.
We normally have antibodies in our blood that repel invaders into our body, such as virus and bacteria microbes. Antinuclear antibodies (ANAs) are unusual antibodies, detectable in the blood, that have the capability of binding to certain structures within the nucleus of the cells. The nucleus is the innermost core within the body's cells and contains the DNA, the primary genetic material. ANAs are found in patients whose immune system may be predisposed to cause inflammation against their own body tissues. Antibodies that are directed against one's own tissues are referred to as auto-antibodies. The propensity for the immune system to work against its own body is referred to as autoimmunity. ANAs indicate the possible presence of autoimmunity and provide, therefore, an indication for doctors to consider the possibility of autoimmune illness.

Antinuclear Antibody: Peripheral Pattern
The ANA test was designed by Dr. George Friou in 1957. The ANA test is performed using a blood sample. The antibodies in the serum of the blood are exposed in the laboratory to cells. It is then determined whether or not antibodies are present that react to various parts of the nucleus of cells. Thus, the term anti-"nuclear" antibody. Fluorescence techniques are frequently used to actually detect the antibodies in the cells, thus ANA testing is sometimes referred to as fluorescent antinuclear antibody test (FANA). The ANA test is a sensitive screening test used to detect autoimmune diseases.
After more than 40 yr, this method is still used as a screening technique, although the employed substrate has evolved from organ tissue to cultured cells. Since the molecular characterization of (most) antigens, other techniques, such as enzyme‐linked immunosorbent assay (ELISA) and immunoblotting, have been developed that allow the precise identification of many ANA specificities. The more precise characterization of the involved antigens has also taught us that some ANA actually react with antigens that do not predominantly occur in the nucleus, but more in the cytoplasm or on the membrane of the cell. Yet, the term ‘antinuclear antibodies’ continues to be used for these antibodies.
Elevated levels of ANA are found in all systemic rheumatic diseases, with sometimes high, sometimes rather loose associations between a particular ANA specificity and a particular rheumatic disease. Therefore, the detection and identification of ANA has gained increasing acceptance by clinicians who use the information to help or confirm a diagnosis and in treatment follow‐up. In this Editorial, we will focus on the role of ANA in the pathogenesis of rheumatic disease, centred around the question ‘Are ANA caused by the disease or the cause of the disease’. First, however, we need to have a closer look at the antigens involved.
Most ANA are directed against nucleic acids or proteins associated with nucleic acids. In systemic lupus erythematosus (SLE), the most predominant antigen is probably the nucleosome.
Nucleosomes form the basic structure of chromatin and have an important function in the compaction of DNA in the nucleus of the cell. A nucleosome consists of dimers of the four core histones, H2A, H2B, H3 and H4, which together form a histone octamer around which 146 bp of DNA are wrapped twice. Two nucleosome subunits are connected via a stretch of linker DNA to which histone H1 is bound. ANA reactive with DNA, especially if measured by a sensitive and specific radioimmunoassay, are considered the hallmark of SLE. Antibodies to histones (and associated ubiquitons) also occur, and more recently, the existence in SLE of nucleosome‐specific antibodies (i.e. recognizing a conformational epitope formed by DNA and histones) has been proven.
Another nuclear particle of importance as an antigen for ANA is the so‐called snRNP (small nuclear ribonucleoprotein) particle. These particles, built from a capped small nuclear RNA molecule (U‐RNA) and a number of polypeptides, have an important role in messenger RNA processing. Mammalian cells contain more than 14 different U‐RNAs and more than 25 different polypeptides have been identified as constituents of the major snRNP particles. Of these proteins, the Sm proteins (BB′, D1/D2/D3, E, F and G) form important antigens for ANA. Other relevant snRNP antigens are the 70 kDa, A and C proteins.
Nuclear antigens also include specificities such as RA‐33, Ku, PCNA, and nuclear enzymes. For a more precise description of these, see [2]. Of the more cytoplasmic antigens, the La/SS‐B protein functions as a termination factor for RNA polymerase III [3]. Transcripts of RNA polymerase III (such as Y‐RNAs, 7S RNA, 5S RNA, tRNA, certain viral RNAs) to which La/SS‐B is bound form the so‐called small cytoplasmic RNP (scRNP) particles. Ro/SS‐A also binds to Y‐RNAs, so Ro/SS‐A and La/SS‐B are often present on the same scRNP particle. In human cells, two different Ro/SS‐A molecules have been identified, designated Ro52 and Ro60. About 30% of both Ro/SS‐A and La/SS‐B is localized in the nucleus of the cell, which explains the nuclear immunofluorescence of anti‐Ro/SS‐A and anti‐La/SS‐B.
Other cytoplasmic antigens of relevance for ANA binding are rRNP (ribosomal RNP) and cytoplasmic enzymes such as aminoacyl tRNA synthetases.

At the time patients are diagnosed as having a rheumatic disease, ANA are generally already present. For certain ANA (e.g. anti‐DNA and anti‐Scl‐70) it has even been shown that their presence in the circulation may precede overt clinical disease by more than 1 yr. So it seems a legitimate question to ask is whether such antibodies indeed mediate the disease features.
Traditionally, ANA were considered to play an active role in disease, since SLE was considered to be an immune complex disease. In this concept, ANA bind to their respective antigens, either in the circulation or in situ, and the resulting immune complexes deposit in the tissues, where subsequent complement activation leads to inflammation and disease features. Especially in the case of anti‐DNA, deposition of DNA/anti‐DNA complexes was supposed to be implicated in the induction of lupus nephritis. In later years, this concept was modified, based on studies that have shown anti‐DNA to interact with tissue structures such as heparan sulphate, the major glycosaminoglycan side chain of the glomerular basement membrane. More recently, such interactions have been shown to be mediated by nucleosomal material.
The concept of anti‐DNA playing a direct role in the pathogenesis of SLE is based on much circumstantial evidence. The various pieces of evidence pointing in the direction of an active role in pathogenesis are:

  • Anti‐DNA fluctuates in time, in close association with exacerbations and remissions of the disease: especially nephritic exacerbations are heralded by an increase in the level of anti‐DNA, while anti‐DNA levels drop steeply during the clinical exacerbation. In fact, upcoming exacerbations can be prevented by treatment of patients on the basis of increasing levels of anti‐DNA.
  • Patients that do not have SLE at the time anti‐DNA is first detected in their circulation generally develop SLE within the next 5 yr.
  • Antibodies to DNA can be eluted from affected kidneys.
  • Perfusion of rat kidneys with histones, DNA and antibodies to DNA leads to the binding of anti‐DNA to the glomerular basement membrane. Initially thought to be based on anti‐DNA cross‐reactivity, we now know this binding is mediated by nucleosomes.

Taken together, these pieces of evidence indicate that anti‐DNA is directly implicated in the induction and propagation of inflammatory reactions in affected tissues.
In longitudinal studies in patients with SLE or Sjögren's syndrome, no relationship between serum levels of anti‐Ro/SS‐A and anti‐La/SS‐B antibodies and clinical symptoms and/or disease exacerbations could be demonstrated. In contrast, in the same patients (SLE group only) anti‐DNA activity correlated very well with disease activity. These findings also implicate that anti‐Ro/SS‐A and anti‐La/SS‐B responses are regulated independently from anti‐DNA. Anti‐Ro/SS‐A and anti‐La/SS‐B responses generally correlate with one another, implicating a central role for scRNP particles in eliciting and sustaining the anti‐Ro/SS‐A and anti‐La/SS‐B responses. Yet, the absence of a correlation with disease activity defies a crucial role in the pathogenesis of the disease.
An exception to this may be found in the relationship between the presence of anti‐Ro/SS‐A (and anti‐La/SS‐B) and the occurrence of isolated congenital heart block (CHB) in their offspring. ANA pass the placenta and remain in the circulation of the child for several weeks after birth. The presence of ANA in the fetal circulation may give rise to neonatal lupus syndrome in the child, and, in some cases the presence of anti‐Ro/SS‐A (and anti‐La/SS‐B) leads to CHB. Anti‐Ro/SS‐A (present in up to 98% of mothers of CHB patients) was reported to form the largest single risk factor for the development of CHB in the child. Yet, the reported risk of having a child with CHB for anti‐Ro/SS‐A‐positive mothers is only about 5%. This risk increases to about 20% in subsequent pregnancies. It is of interest that when (non‐rheumatic) mothers of children with isolated CHB were studied in the setting of the cardiology department of a children's hospital, all mothers were found to have anti‐Ro/SS‐A (and most of the time also anti‐La/SS‐B) in their circulation. Regarding the mechanism of induction of CHB, it has been suggested that anti‐Ro/SS‐A and/or anti‐La/SS‐B react with their corresponding (auto)antigens expressed on the surface of cells of the fetal cardiac conduction system. Indeed, anti‐Ro/SS‐A has been eluted from affected fetal heart tissue. Other proposed mechanisms include cross‐reactivity (of anti‐La/SS‐B) with fetal laminin, or the existence of a special fetal form of Ro/SS‐A (alternative splicing).
These studies indicate that anti‐Ro/SS‐A and maybe anti‐La/SS‐B are indeed involved in the pathogenesis of CHB. Yet, in adults, the antibodies seem to be caused by ‘disease’ only: they are also found in apparently healthy women (mothers of CHB children) and in rheumatic patients do not correlate with disease features.
A correlation between SLE disease exacerbations and levels of anti‐U1‐RNA in the circulation has been demonstrated by Hoet et al.; yet, whether this implies a direct role of this antibody specificity in the pathogenesis of SLE remains to be elucidated.
Of the other ANA specificities, some were found to correlate weakly with disease features (or severity), but no overt roles in the pathogenesis of rheumatic diseases were shown. The absence of disease correlations was recently extended to anticardiolipin (including anti‐beta2‐glycoprotein I and antiprothrombin) antibodies in patients with SLE or the antiphospholipid syndrome.
As discussed above, certain ANA specificities seem to be directly involved in the pathogenesis of disease (e.g. anti‐DNA, anti‐Ro/SS‐A and maybe anti‐U1‐RNA), yet most ANA seem not. This would suggest these ANA to be caused by disease rather than to be the cause of disease. Actually, even though some ANA play a direct role in disease features, this still does not explain their existence. This brings us to the question ‘What causes ANA production’.
It is the current view that autoantigens themselves drive the autoimmune response against them. In this view, nucleosomes are probably the most relevant autoantigens for the genesis of antibodies to nucleosomes, histones and DNA. T cells with nucleosome specificity have been identified in human and murine lupus; these T cells may not only be involved in the induction of antinucleosome antibodies, but also of anti‐DNA antibodies. Burlingame et al. have shown that antinucleosome antibodies precede anti‐DNA in time in MRL/lpr mice. In SLE, increased levels of nucleosomes have been identified in the circulation. Our own results (to be published) even show nucleosome levels to be inversely related to anti‐DNA levels.
With regard to ANA such as anti‐Sm, anti‐U1‐RNP/U1‐RNA, anti‐Ro/SS‐A and anti‐La/SS‐B, the snRNP and scRNP particles might be the antigens involved. A possible clue to the question how nuclear antigens get exposed to the immune system comes from work by Casciola‐Rosen et al., who reported the presence of nucleosomes and other nuclear antigens in surface blebs of cells dying from apoptosis. Indeed, apoptosis has in recent years been implicated in autoimmune disease. Several hypotheses regarding the relationship between apoptosis and autoimmunity have evolved from these studies:

  • A defect in apoptosis may lead to incomplete elimination of autoreactive T cells in the thymus. In MRL/lpr mice both anti‐DNA production and resulting SLE‐like disease are brought about by a defect in CD95 (Fas), a receptor implicated in apoptosis. So far, defects in Fas or its ligand have not been found to be involved in autoimmune disease in man.
  • Prolonged increased apoptosis (due to a combination of inherited traits and environmental factors) may lead to increased levels of apoptotic cells, blebs, or autoantigens in the circulation. Indications of increased apoptosis in SLE have been found in the increased levels of soluble Fas; these levels are the highest in patients bound to develop an exacerbation. Also, as mentioned above, serum nucleosome levels inversely correlate with disease exacerbations.
  • Prolonged increased levels of autoantigens in the circulation may, on the other hand, also result from improper elimination of apoptotic material; e.g. a defect in clearance mechanisms could lead to autoimmunization. Inherited deficiencies of early factors of the complement system often present with SLE. Early complement factors are important for the clearance of immune complexes, but also play a role in the clearance of apoptotic cells. Indeed, mice made deficient for C1q by gene targeting develop ANA and glomerulonephritis.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid, binds specifically to chromatin under physiological conditions by displacement of histone H1. This leads to solubilization of chromatin, which is otherwise insoluble in plasma. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in the clearance of chromatin exposed by cell death. Recently, it was shown that mice with targeted deletion of the SAP gene spontaneously develop ANA and severe glomerulonephritis. These findings indicate that SAP, mediating the clearance of nuclear material, prevents the formation of pathogenic autoantibodies against chromatin and DNA.
(4) Autoantigens present in blebs of apoptotic cells are very sensitive to oxidative challenge or other modifications. This may lead to modifications in the autoantigens, rendering them immunogenic to the autologous immune system.
Due to defects in apoptosis itself or to defects in clearance mechanisms involved with the elimination of apoptotic material, nuclear antigens may become exposed to the immune system and become antigenic. These defects alone may indeed lead to autoimmune disease, such as in C1q‐deficient patients with SLE, or may predispose for autoimmune disease. In the latter case, environmental factors such as (viral) infections may then be the trigger that launches the autoimmunity, and, following from that, autoimmune disease, in such persons.
Once the autoantibodies are present in a person, some play a direct role in the pathogenesis of the disease (e.g. anti‐DNA in SLE and anti‐Ro/SS‐A in CHB), but most just seem to be the result of the disease and, as such, be considered epiphenomena of the disease.
Types of Antibodies
In order to understand the ANA (antinuclear antibody) test, it is first important to understand different types of antibodies.

  • Antibodies are proteins, produced by white blood cells, which normally circulate in the blood to defend against foreign invaders such as bacteria, viruses, and toxins.
  • Autoantibodies, instead of acting against foreign invaders as normal antibodies do, attack the body's own cells.
  • Antinuclear antibodies are a unique group of autoantibodies that have the ability to attack structures in the nucleus of cells. The nucleus of a cell contains genetic material referred to as DNA (deoxyribonucleic acid).

There is an ANA (antinuclear antibody) test which can be performed on a patient's blood sample as part of the diagnostic process to detect certain autoimmune diseases.

When is an ANA test ordered? 
The ANA test is ordered when a patient shows signs and symptoms that are associated with SLE or another autoimmune disorder. It may also be ordered when a patient has been diagnosed with an autoimmune disorder and the doctor suspects that the patient may have developed an additional autoimmune disorder. Patients with autoimmune disorders can have a wide variety of symptoms such as low-grade fever, joint pain, fatigue, and/or unexplained rashes that may change over time.
The ANA test is ordered to help screen for autoimmune disorders and is most often used as one of the tests to diagnose systemic lupus erythematosus (SLE). Depending on the patient’s symptoms and the suspected diagnosis, ANA may be ordered along with one or more other autoantibody tests. Other laboratory tests associated with presence of inflammation, such as erythrocyte sedimentation rate (ESR) and/or C-reactive protein (CRP) may also be ordered. ANA may be followed by additional tests that are considered subsets of the general ANA test and that are used in conjunction with the patient’s clinical history to help rule out a diagnosis of other autoimmune disorders.
An ANA test detects antinuclear antibodies in your blood. Your immune system normally makes antibodies to help you fight infection. In contrast, antinuclear antibodies often attack your body's own tissues — specifically targeting each cell's nucleus.
In most cases, a positive ANA test indicates that your immune system has launched a misdirected attack on your own tissue — in other words, an autoimmune reaction. But some people have positive ANA tests even when they're healthy.
Your doctor may order an ANA test if he or she suspects you have an autoimmune disease such as lupus, rheumatoid arthritis or scleroderma.
Many rheumatic diseases have similar signs and symptoms — joint pain, fatigue and fever. While an ANA test can't confirm a specific diagnosis, it can rule out some possible diseases. And if the ANA test is positive, your blood can be tested for the presence of particular antinuclear antibodies, some of which are specific to certain diseases.
If your blood sample is being used only for an ANA test, you can eat and drink normally before the test. If your blood sample will be used for additional tests, you may need to fast for a certain amount of time before the test. Your doctor will give you specific instructions.

Certain drugs affect the accuracy of the test, so bring your doctor a list of all the medications you take.
For an ANA test, a member of your health care team takes a sample of blood by inserting a needle into a vein in your arm. The blood sample is sent to a lab for analysis. You can return to your usual activities immediately.
The ANA test is ordered to help screen for autoimmune disorders and is most often used as one of the tests to diagnose systemic lupus erythematosus (SLE). Depending on the patient's symptoms and the suspected diagnosis, ANA may be ordered along with one or more other autoantibody tests. Other laboratory tests associated with presence of inflammation, such as erythrocyte sedimentation rate (ESR) and/or C-reactive protein (CRP) may also be ordered. ANA may be followed by additional tests that are considered subsets of the general ANA test and that are used in conjunction with the patient's clinical history to help rule out a diagnosis of other autoimmune disorders.
What are autoimmune diseases?
Autoimmune diseases are conditions in which there is a disorder of the immune system characterized by the abnormal production of antibodies (auto-antibodies) directed against the tissues of the body. Autoimmune diseases typically feature inflammation of various tissues of the body. ANAs are found in patients with a number of different autoimmune diseases, such as systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, polymyositis, scleroderma, Hashimoto's thyroiditis, juvenile diabetes mellitus, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, and pulmonary fibrosis. ANAs can also be found in patients with conditions that are not considered classic autoimmune diseases, such as chronic infections and cancer.
What other conditions cause ANAs to be produced?
ANAs can be produced in patients with infections (virus or bacteria), lung diseases (primary pulmonary fibrosis, pulmonary hypertension), gastrointestinal diseases (ulcerative colitis, Crohn's disease, primary biliary cirrhosis, alcoholic liver disease), hormonal diseases (Hashimoto's autoimmune thyroiditis, Grave's disease), blood diseases (idiopathic thrombocytopenic purpura, hemolytic anemia), cancers (melanoma, breast, lung, kidney, ovarian and others), skin diseases (psoriasis, pemphigus), as well as in the elderly and those people with a family history of rheumatic diseases.
The ANA test is ordered when someone shows signs and symptoms that are associated with SLE or another autoimmune disorder. It may also be ordered when a person has been diagnosed with an autoimmune disorder and the doctor suspects that she may have developed an additional autoimmune disorder. Those with autoimmune disorders can have a wide variety of symptoms such as low-grade fever, joint pain, fatigue, and/or unexplained rashes that may change over time.

What does the ANA test result mean? 
ANA tests are performed using different assays (indirect immunofluorescence microscopy or by enzyme-linked immunoabsorbant assay - ELISA) and results are reported as a titer with a particular type of immunofluroscence pattern (when positive). Low-level titers are considered negative, while increased titers, such as 1:320, are positive and indicate an elevated concentration of antinuclear antibodies.
ANA shows up on indirect immunofluorescence as fluorescent patterns in cells that are fixed to a slide that is evaluated under a microscope. Different patterns are associated with a variety of autoimmune disorders. Some of the more common patterns include:
• Homogenous (diffuse) - associated with SLE and mixed connective tissue disease
• Speckled - associated with SLE, Sjogren’s syndrome, scleroderma, polymyositis, rheumatoid arthritis,    and mixed connective tissue disease
• Nucleolar - associated with scleroderma and polymyositis
• Outline pattern (peripheral) -associated with SLE
An example of a positive result might be: “Positive at 1:320 dilution with a homogenous pattern.”
A positive ANA test result may suggest an autoimmune disease, but further specific testing is required to assist in making a final diagnosis. ANA test results can be positive in people without any known autoimmune disease. While this is not common, the frequency of a false positive ANA result increases as people get older.

Also, ANA may become positive before signs and symptoms of an autoimmune disease develop, so it may take time to tell the meaning of a positive ANA in a person who does not have symptoms. Most positive ANA results don't have significance, so physicians should reassure their patients but should also still be vigilant for development of signs and symptoms that might suggest an autoimmune disease.
About 95% of SLE patients have a positive ANA test result. If a patient also has symptoms of SLE, such as arthritis, a rash, and autoimmune thrombocytopenia, then he probably has SLE. In cases such as these, a positive ANA result can be useful to support SLE diagnosis. Two subset tests for specific types of autoantibodies, such as anti-dsDNA and anti-SM, may be ordered to help confirm that the condition is SLE.
A positive ANA can also mean that the patient has drug-induced lupus. This condition is associated with the development of autoantibodies to histones, which are water soluable proteins rich in the amino acids lysine and arginine. An anti-histone test may be ordered to support the diagnosis of drug-induced lupus.
Other conditions in which a positive ANA test result may be seen include:
• Sjögren’s syndrome: Between 40% and 70% of patients with this condition have a positive ANA test result. While this finding supports the diagnosis, a negative result does not rule it out. The doctor may want to test for two subsets of ANA: Anti-SS-A (Ro) and Anti-SS-B (La). The frequency of autoantibodies to SSA in patients with Sjögren’s can be 90% or greater.
• Scleroderma: About 60% to 90% of patients with scleroderma have a positive ANA finding. In patients who may have this condition, ANA subset tests can help distinguished two forms of the disease, limited versus diffuse. The diffuse form is more severe. Limited disease is most closely associated with the anticentromere pattern of ANA staining (and the anticentromere test), while the diffuse form is associated with autoantibodies to the anti–Scl-70.
• A positive result on the ANA also may show up in patients with Raynaud’s disease, rheumatoid arthritis, dermatomyositis, mixed connective tissue disease, and other autoimmune conditions.
A doctor must rely on test results, clinical symptoms, and the patient’s history for diagnosis. Because symptoms may come and go, it may take months or years to show a pattern that might suggest SLE or any of the other autoimmune diseases.
A negative ANA result makes SLE an unlikely diagnosis. It usually is not necessary to immediately repeat a negative ANA test; however, due to the episodic nature of autoimmune diseases, it may be worthwhile to repeat the ANA test at a future date.
Aside from rare cases, further autoantibody (subset) testing is not necessary if a patient has a negative ANA result.
The presence of any antinuclear antibodies is a positive test result. But having a positive result doesn't mean you have a disease. Many people with no disease have positive ANA tests — particularly women older than 65.
Mononucleosis is one type of infection that has been associated with the development of antinuclear antibodies. Some blood pressure lowering drugs and certain anti-seizure medications may trigger antinuclear antibody formation as well.
If your doctor suspects you have an autoimmune disease, he or she is likely to order a number of tests. The result of your ANA test is one piece of information your doctor can use to help determine the cause of your signs and symptoms.

Positive ANA Result Explained
ANAs are found in patients who have various autoimmune diseases, but not only autoimmune diseases. ANAs can be found also in patients with infections, cancer, lung diseases, gastrointestinal diseases, hormonal diseases, blood diseases, skin diseases, and in elderly people or people with a family history of rheumatic disease. ANAs are actually found in about 5% of the normal population.
The ANA results are just one factor in diagnosing, and must be considered together with the patient's clinical symptoms and other diagnostic tests. Medical history also plays a role because some prescription drugs can cause "drug-induced ANAs".
Incidence of ANA in Various Diseases
Statistically speaking the incidence of positive ANA (in percent) per conditon is:

  • Systemic lupus erythematosus (lupus or SLE) - over 95%
  • Progressive systemic sclerosis (scleroderma) - 60-90%
  • Rheumatoid Arthritis - 25-30%
  • Sjogren's syndrome - 40-70%
  • Felty's syndrome - 100%
  • Juvenile arthritis - 15-30%

Subsets of the ANA (antinuclear antibody) test are sometimes used to determine the specific autoimmune disease. For this purpose, a doctor may order anti-dsDNA, anti-Sm, Sjogren's syndrome antigens(SSA, SSB), Scl-70 antibodies, anti-centromere, anti-histone, and anti-RN.
The ANA (antinuclear antibody) test is complex, but the results (positive or negative, titer, pattern) and possible subset test results can give physicians valuable diagnostic information.
ANA tests are performed using different assays (indirect immunofluorescence microscopy or by enzyme-linked immunoabsorbant assay, ELISA) and results are reported as a titer, often with a particular type of immunofluroscence pattern (when positive). Low-level titers are considered negative, while increased titers, such as 1:320, are positive, indicating an elevated concentration of antinuclear antibodies.
ANA shows up on indirect immunofluorescence as fluorescent patterns in cells that are fixed to a slide that is evaluated under a microscope. Different patterns have been associated with a variety of autoimmune disorders, although overlap may occur. Some of the more common patterns include:
Homogenous (diffuse) - associated with SLE and mixed connective tissue disease
Speckled - associated with SLE, Sjogren syndrome, scleroderma, polymyositis, rheumatoid arthritis, and mixed connective tissue disease
Nucleolar - associated with scleroderma and polymyositis
Centromere pattern (peripheral) - associated with scleroderma and CREST (Calcinosis, Raynaud's syndrome, Esophogeal dysmotility, Sclerodactyly, Telangiectasia)
An example of a positive result might be: "Positive at 1:320 dilution with a homogenous pattern."
A positive ANA test result may suggest an autoimmune disease, but further specific testing is required to assist in making a final diagnosis. ANA test results can be positive in people without any known autoimmune disease. While this is not common, the frequency of a false positive ANA result increases as people get older.
Also, ANA may become positive before signs and symptoms of an autoimmune disease develop, so it may take time to tell the meaning of a positive ANA in a person who does not have symptoms. Most positive ANA results don't have significance, so physicians should reassure their patients but should also still be vigilant for development of signs and symptoms that might suggest an autoimmune disease.
About 95% of those with SLE have a positive ANA test result. If someone also has symptoms of SLE, such as arthritis, a rash, and autoimmune thrombocytopenia, then she probably has SLE. In cases such as these, a positive ANA result can be useful to support SLE diagnosis. Two subset tests for specific types of autoantibodies, such as anti-dsDNA and anti-SM, may be ordered (often as an ENA panel) to help confirm that the condition is SLE.
A positive ANA can also mean that the person has drug-induced lupus. This condition is associated with the development of autoantibodies to histones, which are water-soluble proteins rich in the amino acids lysine and arginine. An anti-histone test may be ordered to support the diagnosis of drug-induced lupus.
A doctor must rely on test results, clinical symptoms, and the person's history for diagnosis. Because symptoms may come and go, it may take months or years to show a pattern that might suggest SLE or any of the other autoimmune diseases.
A negative ANA result makes SLE an unlikely diagnosis. It usually is not necessary to immediately repeat a negative ANA test; however, due to the episodic nature of autoimmune diseases, it may be worthwhile to repeat the ANA test at a future date if symptoms persist.
Aside from rare cases, further autoantibody (subset) testing is not necessary if a person has a negative ANA result.

Antinuclear Antibody Testing Dilemmas:
Does High Throughput Trump Sensitivity?
Autoantibodies have been used as a biomarker for systemic autoimmune rheumatic diseases (SRDs) for more than a half-century and have found a solid place as a tool in diagnosing conditions such as systemic lupus erythematosus, scleroderma, and Sjögren syndrome. Recently, however, evolving technologies and inadequate understanding of the limitations of various methods have led antinuclear antibody (ANA) results to be used inappropriately as a screening test for SRDs. Given this challenge, laboratorians have a responsibility to know the ins-and-outs of their methods and educate physicians to make the best use of ANA testing, according to experts.
“Many primary care practitioners have an imperfect understanding of the diagnostic criteria for autoimmune disorders and the pitfalls that go along with ANA screening testing. There is a problem, a big problem, of false, clinically irrelevant positives in a large minority of normals. This is well-appreciated by rheumatologists, but generalists really are not that aware of it,” said John L. Carey, MD, vice-chair of pathology and laboratory medicine at Henry Ford Health System in Detroit. “This ocean of patients who do not have systemic rheumatic disease, yet test positive, destroys the positive predictive value of ANA tests. Unfortunately, this is not appreciated by clinicians and it really challenges the lab to correct a false impression created by clinically irrelevant positive tests.”

The Pros and Cons of Immunofluorescence
False positives are not the only issue labs face in educating physicians about ANA testing. Both old and new testing technologies are evolving, and evidence is emerging that could help improve the specificity of established methods. Indirect immunofluorescence (IIF) has been used since the 1950s to detect ANAs, initially using rodent organs and now, most commonly, human epithelioid laryngeal cancer cell (HEp-2) cell lines as substrates. IIF offers the advantages of providing both ANA pattern and titer information, and detecting in excess of 100 autoantibodies. The method has very good sensitivity for certain SRDs, such as lupus (85%), mixed connective tissue disease (100%), and drug-induced lupus (100%).
However, IIF has some downsides as well. It suffers from being a non-standardized manual test and having subjective result interpretation and low reproducibility. This time-consuming, low throughput test also produces its share of false negatives for certain autoantibodies, including Jo-1 (30%), Ribosomal P (40%), and proliferating cell nuclear antigens (30%). At any given time, IIF misses about 15% of lupus patients, too, but when established patients are followed over time, approximately 97–98% will have positive IIF results, according to Carey.
On the flip side, as many as 13% of healthy individuals will have positive ANA IIF results at a 1:80 dilution. Though higher titers generally are thought to be more clinically significant and indicative of SRD, evidence suggests that beyond a threshold of 1:80 or 1:160, ANA titer “has little bearing on diagnosis or disease activity,” according to Marvin Fritzler, MD, PhD, professor of medicine at the University of Calgary in Alberta, Canada.
What’s in the Pattern?
Given the sometimes limited information provided by titers, clinicians also use the staining pattern for diagnostic clues. Several IIF patterns have been linked to specific antigens and SRDs. For instance, a mitotic apparatus pattern is associated with nuclear mitotic apparatus 1, which is present in lupus and Sjögren syndrome. However, the staining pattern is not always specific for ANA or disease. As an example, a speckled pattern has been associated with certain ANAs like SS-A/Ro, but it also is found in patients with a variety of SRDs, as well as in healthy individuals.
Even with the limitations of both titer and staining patterns, some researchers are looking closer at staining pattern as a way to discriminate healthy individuals from those with SRDs. In a recent study comparing healthy subjects to patients with SRDs, Brazilian researchers found distinct pattern profiles between the two cohorts and suggested that pattern is a “critical parameter” for interpreting positive ANA IIF results (Arthritis Rheum 2011;63:191–200). The nuclear dense fine speckled pattern occurred only in healthy individuals, and at a moderate titer of 1:640 in nearly two-thirds. In contrast, nuclear course speckled, nuclear homogeneous, and nuclear centromeric patterns were found only in patients with SRDs. The nuclear fine speckled pattern was common to both groups, but tended to be of high titer in patients with SRDs and low titer for healthy individuals.
“Our daily practice suggested that the ANA pattern might play a role in the interpretation of a positive ANA test. However, we were surprised by the extent with which this parameter may guide the clinical significance of a positive ANA test,” said the study’s senior author, Luis E. C. Andrade, MD, PhD, associate professor of medicine and head of rheumatology at Universidade Federal de São Paulo, Brazil. “Our results imply that certain ANA patterns, such as the nuclear homogeneous and the nuclear coarse speckled pattern, should direct a thorough clinical and laboratory investigation to unravel a systemic autoimmune disease, especially within the spectrum of systemic lupus erythematosus. Even if no abnormality is found in an initial screening, it may be wise to follow such patients for a while because they may be undergoing an incipient stage of the disease.”
While Andrade and his coauthors called for further research to test the reproducibility of their findings, he suggested that labs could readily implement this approach to pattern recognition. “The analyst in an ordinary clinical laboratory should be able to recognize the ANA patterns that are clinically relevant and those that are most probably observed in individuals with no apparent autoimmune disease,” he commented.
In an accompanying editorial, Fritzler agreed that “this study may provide the leading edge of understanding which autoantibodies rule out the diagnosis of systemic ARD.” However, he went on to caution that as promising as the findings may be, “interlaboratory exchange of sera and the establishment of a validated reference standard producing the nuclear dense fine speckled IIF pattern are desperately needed before these observations can be put into wide clinical laboratory practice.”
Technological, Standardization Advances
Recently, some manufacturers have taken steps to automate both the analytical and pre-analytic components of IIF ANA testing, including using automatic fluorescent image analysis to provide a virtual titer, thereby eliminating the process of manually staining a series of diluted samples. At least one system simultaneously reads multiple slides and provides basic positive or negative results and pattern recognition.
Recognition of the variability of IIF and other types of autoantibody testing methods lead in the early 1980s to formation of the Autoantibody Standardization Committee in Rheumatic and Related Diseases under the auspices of the International Union of Immunological Societies, the World Health Organization, Arthritis Foundation, and the Centers for Disease Control and Prevention (CDC). In the intervening decades, the committee has overseen development of reference sera for 16 ANAs which are available through CDC to research and diagnostic laboratories and companies developing autoantibody diagnostic kits. Andrade, who chairs the committee, indicated that the group is on the verge of releasing a standard for citrullinated peptides and is in the process of establishing standards for antibodies to beta-2 glycoprotein 1.
Other standardization efforts are underway as well. For instance, the European Autoimmunity Standardization Initiative was founded in 2002 with the goal of improving diagnostics in chronic rheumatic disorders by strengthening the collaboration between clinical and laboratory scientists responsible for autoimmune diagnostics.
The Move to Higher Throughput
Even as technology, the evidence base, and standardization efforts coincide to improve the performance of IIF and other traditional assays based on hemagglutination reactions and immunodiffusion, these methods are being given a run for their money by enzyme immunoassays (EIA) and other solid phase methods like microarrays and bead-based assays. The latter have come on strong in the past 15 years, and may account for more than half the testing currently performed in the U.S., since many high-volume labs use the tests, according to David Keren, MD, medical director of Warde Medical Laboratory in Ann Arbor, Mich.
These methods offer some distinct advantages in an era of belt-tightening in healthcare. Automated and faster than IIF, they run on platforms that can be used to perform other tests and are easier for lab staff to maintain proficiency on. The quality of these tests has improved over time, and College of American Pathologists surveys show that both EIA and multiplex tests perform well in comparison to IIF, according to Keren. But they have their own set of challenges.
“The solid-phase assays such as bead and EIA assays include relatively few antigens in comparison to intact HEp-2 cells. They also don’t provide information such as patterns of reactivity and strength of titer, which rheumatologists have found useful in evaluating their patients,” explained Keren. “Further, manufacturers of solid phase assays use different combinations of antigens and even within one manufacturer, one must compare lot-to-lot variability.”
What’s it Missing?
One analysis of commercial multiplexed ANA screening kits found that they used a range of six to 15 antigen combinations (Lupus 2005;15:412–421). Depending on how they’re made, some solid-phase assays contain, in addition to specific antigens, an extract of HEp-2 cells that enables detection of a broader range of antigens. While these assays generally perform well, depending on a patient’s SRD and the type of autoantibodies he or she has, the assays will do better or worse. For instance, a recent analysis of scleroderma patients found that only 51% tested positive by multiplex assay versus 91% by IIF (Clin Rheumatol 2011 DOI 10.1007/s10067-011-1766–6). Although there was good agreement between the methods in detecting Sc170, RNP, and centromere antibodies, the multiplex assays did not detect several autoantibodies of importance in scleroderma.
“The test performs well in what it’s been designed to do, which is to detect those specific antibodies that have been put into the assay. The problem is, for certain subsets of patients, it has a high false-negative rate,” explained the study’s lead author, Victoria Shanmugam, MD, an assistant professor of medicine at Georgetown University in Washington, D.C. In the case of scleroderma, one of the missed autoantibodies, RNA polymerase III, is associated with poor outcomes, so the insensitivity of some assays in picking it up is a concern.
“Those patients are at high risk of renal crisis and can have abrupt onset of disease, presenting initially with renal crisis and not having other clinical signs like skin thickening. So the emergency physician or internist may not consider scleroderma as an underlying etiology because they’re seeing a negative ANA. That potentially could lead the patient to not being treated appropriately.”

Is there anything else I should know? 
Some drugs and infections as well as other conditions mentioned above can give a false positive result for the ANA test.
About 3% - 5% of Caucasians may be positive for ANA and it may reach as high as 10% - 37% in healthy individuals over the age of 65.
Some medications may bring on a condition that includes SLE symptoms, called drug-induced lupus. When the drugs are stopped, the symptoms usually go away. Although many medications have been reported to cause drug-induced lupus, those most closely associated with this syndrome include hydralazine, isoniazid, procainamide, and several anticonvulsants.
To perform the ANA (antinuclear antibody) test, sometimes called FANA (fluorescent antinuclear antibody test), a blood sample is drawn from the patient and sent to the lab for testing.
Serum from the patient's blood specimen is added to microscope slides which have commerically prepared cells on the slide surface. If the patient's serum contains antinuclear antibodies (ANA), they bind to the cells (specifically the nuclei of the cells) on the slide.
A second antibody, commercially tagged with a fluorescent dye, is added to the mix of patient's serum and commercially prepared cells on the slide. The second (fluorescent) antibody attaches to the serum antibodies and cells which have bound together. When viewed under an ultraviolet microscope, antinuclear antibodies appear as fluorescent cells.
If fluorescent cells are observed, the ANA (antinuclear antibody) test is considered positive.
If fluorescent cells are not observed, the ANA (antinuclear antibody) test is considered negative.
ANA Titer
A titer is determined by repeating the positive test with serial dilutions until the test yields a negative result. The last dilution which yields a positive result (fluorescence) is the titer which gets reported. For example, if a titer performed for a positive ANA test is:
1:10 positive
1:20 positive
1:40 positive
1:80 positive
1:160 positive
1:320 negative
The reported titer in our example is 1:160.
Parts of an ANA Report
An ANA report has three parts:

  • positive or negative
  • if positive, a titer is determined and reported
  • the pattern of fluorescence is reported

Significance of ANA Pattern
ANA titers and patterns can vary between laboratory testing sites, perhaps because of variation in methodology used. These are the commonly recognized patterns:

  • Homogeneous - total nuclear fluorescence due to antibody directed against nucleoprotein. Common in SLE (lupus).
  • Peripheral - fluorescence occurs at edges of nucleus in a shaggy appearance. Anti-DNA antibodies cause this pattern. Also common in SLE (lupus).
  • Speckled - results from antibody directed against different nuclear antigens.
  • Nucleolar - results from antibody directed against a specific RNA configuration of the nucleolus or antibody specific for proteins necessary for maturation of nucleolar RNA. Seen in patients with systemic sclerosis.

A number of drugs and some infections (such as chronic non-viral hepatitis, primary biliary cirrhosis) as well as other conditions mentioned above can give a false positive result for the ANA test.
About 3% - 5% of Caucasians may be positive for ANA and it may reach as high as 10% - 37% in healthy individuals over the age of 65.
A number of medications may bring on a condition that includes SLE symptoms, called drug-induced lupus. When the drugs are stopped, the symptoms usually go away. Although many medications have been reported to cause drug-induced lupus, those most closely associated with this syndrome include hydralazine, isoniazid, procainamide, and several anticonvulsants.
Though some laboratories may use an immunoassay to test for ANA, indirect fluorescent antibody (IFA) is still considered the gold standard. Often, laboratories will screen using immunoassay and confirm positive or equivocal results using IFA.

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