The antinuclear antibody (ANA) test is widely used as a serological marker of autoimmune disease. Antinuclear antibodies are immunoglobulins or antibodies that bind to one or more antigens expressed within the nucleus of human cells. Used selectively, the ANA test can be a useful laboratory tool to help confirm or exclude the diagnosis of systemic rheumatic disease. However, the relatively high prevalence of ANAs in other inflammatory conditions, as well as healthy individuals, can make a positive result difficult to interpret.
How is the test performed?
Although many methods are available for ANA detection, the indirect immunofluorescence antinuclear antibody test (IF-ANA) and enzyme immunoassay (EIA)/enzyme linked immunosorbent assay (ELISA) are commonly used.
Indirect IF-ANA involves incubating patient serum on a slide covered with a monolayer of cells from a malignant human epithelial cell line. These malignant cells are ideal for the ANA test as they have large nuclei in different stages of the cell cycle. The slides are washed and any remaining antibodies bound to cell nuclei are then visualised using a detection antibody binding human immunoglobulin which has been conjugated to a fluorescent tag. The detection antibody will then be visible using a fluorescence microscope if there is immunoglobulin from the patient serum that has bound to the cells on the slide.
In ELISAs wells are coated with antigens from cell nuclei. Serum is incubated in the wells and antibodies binding to the antigens are then detected using a detection antibody which has been conjugated with an enzyme tag. Antibody levels may be quantified by the amount of colour change of a substrate by the enzyme tag.
How is the test reported?
The results of ANA testing are reported in two components: the quantity of ANA in the serum (intensity) and, when the ANA is positive, the pattern of antibody binding to the nucleus (staining pattern).
The quantitation of an ANA is most commonly reported as a titre, reflecting the final step in a series of two-fold dilutions at which the ANA remains positive (eg. 1:1 280 for a strongly positive ANA, or 1:160 for a weaker, borderline positive ANA). An alternative method involves reporting the intensity of fluorescence, in international units per millilitre (IU/mL), at a pre-determined dilution. With this method, a result of >7 IU/mL is generally considered positive.
The different staining patterns provide clues to the significance of the ANA and type of rheumatic disease
Low-intensity ANAs are present in up to 40% of healthy individuals.1 To address this issue, most laboratories will set a cut-off for reporting a positive ANA that excludes the majority of these low-intensity and clinically insignificant results. However, at least 5% of the healthy population have a moderate titre ANA that is considered positive; there are relatively higher rates in women and the elderly. If a randomly selected population were to be screened with ANA testing, 50 or more healthy people with a positive ANA would be identified for every one patient with systemic lupus erythematosis (SLE). A pre-existing clinical suspicion of systemic rheumatic disease is critical to enhance the clinical utility of a positive ANA result.2 In the absence of clinical or laboratory markers supporting a diagnosis of rheumatic disease, a positive ANA is seldom useful.
A positive ANA will be seen in a range of conditions where it is not diagnostically helpful. These include non-autoimmune conditions such as chronic infection, viral hepatitis and malignancy, and also some autoimmune conditions such as multiple sclerosis or thyroid disease where the presence or absence of ANA does not play a significant role in diagnosis or prognosis.3
Many patients with autoimmune disease will not have a positive ANA. While the ANA test is highly sensitive for certain rheumatic diseases such as SLE and systemic sclerosis/scleroderma, a negative result doesn’t exclude a wide range of other conditions including rheumatoid arthritis, spondyloarthropathies, idiopathic inflammatory myopathies and vasculitides.
The ANA test is rarely interpreted in isolation, as there is likely to be a broad differential for the clinical presentation of a patient in whom autoimmune rheumatic disease is suspected. As a sensitive, but non-specific, marker of some systemic rheumatic diseases, the ANA test can be performed early in the diagnostic evaluation of patients with suggestive clinical symptoms to better direct further investigations.
In the majority of patients, the ANA test result alone is NOT sufficient to confirm the diagnosis of and characterise a systemic rheumatic disease. If ANA is positive, more specific tests may be performed based on clinical findings and ANA staining patterns. Antibodies directed against particular antigens within the cell nucleus are more strongly associated with particular rheumatic diseases. Therefore, testing for antibodies to these extractable nuclear antigens (ENA) is a useful follow-up test in patients with a positive speckled/peripheral ANA. Antibodies that can be characterised by the ENA test include anti-Sm (highly specific for SLE but only found in less than one-third of patients4), and anti-SSA and anti-SSB (also know as Ro and La respectively, seen in patients with Sjögren’s syndrome and cutaneous lupus, and associated with a 1–2% risk of congenital heart block in foetuses of antibody-positive mothers).5 Additionally, in patients with a homogenous pattern ANA, antibodies against double-stranded DNA can be quantitated by a separate test (anti-dsDNA antibody test). These antibodies are specific for SLE and the level of antibody may fluctuate with disease activity.
The ANA results are often interpreted in conjunction with other investigations selected according to the clinical scenario. The anti-cyclic citrullinated peptide antibody (anti-CCP) test (which has a high specificity for rheumatoid arthritis), full blood count (cytopaenias are a feature of SLE), urinalysis (haematuria and/or proteinuria may be due to renal manifestations of autoimmune disease), serum complement proteins C3 and C4 (low complement can reflect consumption in immune complex-mediated diseases such as SLE) and immunoglobulins (increased due to chronic inflammation, often markedly so with Sjögren’s syndrome) are all potentially useful companion tests when interpreting a positive ANA.
What should a patient be told about the test?
Given the low specificity of ANA for systemic autoimmune disease, it is important to counsel the patient about the limitations of a positive test result, particularly if it is requested with low pre-test probability. A positive ANA is not diagnostic of lupus (or any other autoimmune disease) and most individuals with positive ANA will not develop an autoimmune disease in the subsequent three years if they don’t have any suggestive symptoms at the time of initial testing.2
The ANA test is performed on serum and does not require any special patient preparation or collection. Due to the stability of immunoglobulins, stored serum samples will often be suitable. In patients with positive ANA, stored serum may be available to perform additional testing for ENA and dsDNA antibodies to potentially provide more specific diagnostic information. While Medicare benefits are payable for each of these tests (ANA, ENA and dsDNA), they represent three separate item numbers and therefore episode coning rules may apply.
What do the results mean?
A positive test
The response to a positive test result will depend on the clinical scenario. Most commonly, the ANA test will be requested when there are features of systemic rheumatic disease. Therefore, follow-up testing for ENA antibodies is generally helpful as this might provide some more specific diagnostic information. If SLE is suspected, dsDNA antibody measurement is indicated.
The diagnosis of systemic rheumatic disease is usually based on a number of clinical and laboratory criteria, so a positive ANA could prompt a more targeted review of the clinical history and examination findings, and additional laboratory testing directed at those potential manifestations (see above).
Most positive ANA tests are not associated with systemic rheumatic disease, particularly at low titre. In subspecialty rheumatology clinics it is reported that <10% of patients referred with low to moderate titre positive ANA are ultimately diagnosed with an ANA-associated rheumatic disease.6
There is no absolute cut-off at which a positive ANA is clinically significant, however the higher the ANA titre the more likely it is to be associated with systemic rheumatic disease.6 Therefore, in patients with high titre ANAs (the definition of which may change between laboratories but often >1:1 280) periodic assessment for development of new symptoms is reasonable. While many of these patients will never develop autoimmune disease, a positive ANA may appear several years before clinical symptoms in patients ultimately diagnosed with lupus.7
A negative test
A negative ANA test effectively excludes a diagnosis of ANA associated rheumatic disease such as drug-induced lupus and, in the majority of cases, SLE, systemic sclerosis/scleroderma and mixed connective tissue disease, particularly where there is a low pre-test probability of these conditions. However, while many other autoimmune diseases are associated with a positive ANA, it is a relatively insensitive marker for most of those conditions and therefore they can’t be discounted based purely on negative ANA results (Table 2). These conditions include Sjögren’s syndrome, rheumatoid arthritis, discoid lupus, juvenile idiopathic arthritis (without uveitis) and various forms of vasculitis including giant cell arteritis, polyarteritis nodosa, granulomatosis with polyangiitis (formerly Wegener’s granulomatosis) and eosinophilic granulomatosis with polyangiitis (Churg Strauss syndrome).
Key points
The ANA test is a useful tool for the evaluation of patients with symptoms of systemic rheumatic disease.
ANA testing in patients with a low probability of rheumatic disease is likely to result in high numbers of false positive results.
A positive ANA test is not diagnostic of autoimmune disease and is seen in many non-rheumatic conditions as well as healthy individuals.
High titre ANAs are associated with a higher likelihood of rheumatic disease, but interpretation of their significance requires correlation with clinical symptoms and other investigations.
Reference
The Speckled pattern correlates with Sjogren's Syndrome.
See also: lupus-support.org/LuPUSMB/i...
The term antinuclear antibody (ANA) describes a variety of autoantibodies that react with constituents of cell nuclei including DNA, proteins and ribonucleoproteins1,2. The HEp-2 cell, a native protein array with hundreds of antigens, provides an ideal substrate for the detection of ANA1. The detection of ANA in human serum is an important screening tool for connective tissue diseases, and IIF is the reference method for ANA testing1. Recently, IIF on HEp-2 cells has been replaced in some laboratories with antigen-specific immunoassays and multiplex methods. Due to concerns over false negative results and the lack of transparency to clinicians, the American College of Rheumatology formed a Task Force that concluded that IIF using HEp-2 cells should be the “gold standard” for ANA screening.
See: jove.com/video/51211/anti-n...
Disclaimer: No attempt is made to diagnose or to make any medical judgement. You are advised to seek the advice from your own physician. LUpus Patients Understanding & Support (LUPUS) is not a substitute for your own doctor.
