Connective tissue diseases (CTD) are a group of autoimmune disorders which are characterized by presence of antinuclear antibodies (ANA) in the blood of patients. ANA are a specific class of autoantibodies that have the capability of binding and destroying certain structures within the nucleus of the cells 1. Although lower amounts of these antibodies can be seen in the normal population as well, a spurt in titers is seen in patients of CTD. Not only are these antibodies involved in the disease pathogenesis, but they also constitute the basis for diagnosis and treatment of CTD. Their detection with high sensitivity and specificity is therefore of utmost importance. Various detection methods are in use and there is continuous pouring of newer techniques to facilitate diagnosis and therapeutic monitoring in CTD patients. In this review we have discussed in brief how ANA were discovered and found to be associated with CTD. This article also gives an overview on advancement in various ANA detection methods, their future prospects along with advantages, disadvantages and guidelines for use of these tests.

In 1941, Klemperer, Pollack and Baehr first described systemic lupus erythematosus (SLE) as one of the CTD 2. Then in 1948 Malcom Hargrave, Helen Richmond and the medical resident Robert Morton noted the presence of previously unknown cells in the bone marrow of a patient with SLE. They called these LE cells and described them as mature polymorphonuclear leukocytes which had phagocytosed the liberated nuclear material of another leukocyte 3. This extremely important discovery laid the foundation of research for ANA. Since then, ANA has been divided into specific subtypes based on the nuclear or cytoplasmic component they attack i.e. anti-DNA, anti-histone etc.

Presently the ANA have been categorized in to 2 main groups:

Presence of autoantibodies in the sera of the patient constitutes one of the criteria used for diagnosis of CTD (table 2). Besides clinical diagnosis the ANA subtyping also helps in identifying a specific CTD 22. Although a battery of laboratory tests are available for ANA detection indirect immunofluorescence antinuclear antibody test (IF-ANA) and enzyme immunoassay (EIA)/enzyme linked immunosorbent assay (ELISA) are commonly used in day to day practice. Some of them are considered outdated while others like flowcytometry and recently introduced nanotechnology involving antigen arrays are still in experimental stages.

Different staining patterns are reported which give clues as to the significance of the ANA and type of CTD (table 3, figure 1):

1. Nuclear patterns: homogeneous, speckled (fine and coarse), peripheral/rim, nucleolar, centromeric, PCNA (proliferating cell nuclear antigen), nuclear dots, nuclear membrane, diffuse grainy.

2. Cytoplasmic patterns: speckled, mitochondrial-like, ribosomal-like, Golgi apparatus, lysosomal-like, cytoskeletal filaments (actin, vimentin, cytokeratin)

3. Mitotic patterns: mitotic spindle, centrosomes, NuMA (nuclear mitotic apparatus), midbody, CENP-F (centromere protein)

Among these homogenous, speckled, peripheral and nucleolar patterns are more commonly observed and of clinical importance. With any of these fluorescence patterns intensity of staining with a qualitative scale of values from + to ++++ should also be reported as fluorescence intensity is generally proportional to antibody concentration and predicts the severity of the CTD.

Sera of some patients with SLE may be negative on animal substrates i.e. mouse kidney or rat liver but are positive on human substrate i.e. Hep-2 cell lines 262728. Due to variable sensitivity with the substrate used it is essential to report the type of substrate being used by the lab.

It is directly proportional to antibody concentration and expressed with a quantitative scale of values. Its evaluation is crucial as low titer is less significant than a high titer and may be seen even in healthy individuals. There are many studies which have attempted to determine the optimum screening dilution of sera for ANA testing. A titer of 1:160 is taken as significant for the diagnosis of CTDs in majority of laboratories 2930.

Although IF-ANA test is widely used and considered to be gold standard still the results may sometimes be misinterpreted. As it detects several different antibodies cross-reactions can occur. In up to 3% of the normal population it can give false positive result. Also ANA levels tend to rise when symptoms flare and fall, often being undetectable, when symptoms are mild or patient is in remission. Moreover each CTD has specific antibody associated with it and sometimes it is difficult to specify or categorize an autoantibody 3132. Certain patterns i.e. nucleolar and centromeric are less well defined by IF-ANA tests. The test therefore is mainly used for screening rather than to diagnose a CTD.

There are two types of EIA or ELISA methods currently used for ANA testing. One is called generic assay which detects ANA of broad specificity similar to IF-ANA and other is antigen specific assay that detects ANA and reacts with a single autoantigen i.e. dsDNA, SS-A/Ro, SS-B/La, Scl-70, Sm, Sm/RNP etc. In antigen specific assay multiple antigens are coated on to microtitre plates, usually a combination of SSA/Ro, SSB/La, Sm, and U1-RNP, with many also including Jo-1 and Scl70. This new test is both highly specific and sensitive and substantially decreases the time involved when screening large numbers of patient samples. The test is simple to perform, can be automated and does not require highly trained operators who can recognize microscopic patterns. The EIA/ELISA is therefore becoming the most widely used method not only for routine screening but also for detection of specific ANA. Kits available in the market either utilize extracts of tissue containing various nuclear components or molecules synthesized by recombinant technology. The later may include individual recombinant molecules such as SS-A/Ro, or combinations of molecules which increase the sensitivity of the test. In a recent study, the performance of ELISA test was compared with the "gold standard" IF-ANA test. The agreement that a serum is ANA positive was 87% to 95% when comparing the ELISA and IF-ANA test results 33. The sensitivity of the various ELISAs was 69% to 98% and the specificity ranged between 81% and 98%. These figures were arrived at using sera that were positive at 1:160 by the IF-ANA test. The above comparison figures were much lower for sera with IF-ANA titer of 1:40.

Although the second multicentre European study showed that ELISA methods are improving 34, the recent study by Bizzaro et al suggests that the problem of false positive results in ELISA is still widespread 35. ELISA may miss anti-SSA/Ro even when using the reference sera. This is probably a result of the vigorous antigen preparation methods. Sera that react only with conformational antigens can also miss the presence of antigen. The ELISA techniques have also been found to miss a low titer positive ANA as well as sera with specific ANA. Presently, ELISA tests therefore may be adequate to screen sera only with intermediate to high titers. It remains to be seen from further studies whether the performance of screening ANA tests by ELISA would match that by the fluorescent technique 36.

A positive ANA result in conjunction with clinical findings is diagnostic therefore frequently asked by the clinician in case of suspected CTD. Since different ANA are associated with one or other CTD a systematic approach has to be followed while performing these tests. Therefore initially screening is carried out usually by IF-ANA/ELISA and if positive more specific tests are performed based on clinical findings and IF-ANA staining patterns (table 3).

Autoantibody to dsDNA is specific and diagnostic for SLE and levels are elevated during active disease. Therefore in a case of suspected SLE if homogenous pattern is observed on IF-ANA further tests i.e. CLIF, ELISA, blotting tests etc. may be done to confirm dsDNA. Similarly anti-Sm is highly specific for SLE and needs confirmation by other tests i.e. Blotting etc. but is present in only 10% of SLE cases.

Anti-SSA/Ro antibody although more common in Sjogren's syndrome but can also be found in 30% cases of SLE with cutaneous involvement. Therefore if IF-ANA shows speckled/peripheral pattern further tests i.e. Blotting, MIA are required for detection of anti-SSA/Ro antibody. Clinical significance and detection methods for anti-SSB/La are similar to that for anti-SSA/Ro except that it is less common and may indicate minor course of disease. While presence of these two autoantibodies supports Sjogren's syndrome they are not much needed for diagnosis. Anti-Scl-70 autoantibody found in scleroderma (SS) gives a fine speckled staining pattern on IF-ANA and can be confirmed by immunodiffusion techniques but its detection is also not a necessity for diagnosis.

Antinucleolar antibodies are a group of autoantibodies which give nucleolar staining pattern. Most common of these are anti-PM-Scl, anti-RNA polymerase I-III and anti-U3-RNP (antifibrillarin). Although seen in scleroderma and polymyositis (PM) their detection is also not widely practiced 24.

A protocol generally followed by the clinicians and step by step approach to detect all these autoantibodies has been described in figure 2. A summary of certain other guidelines 2461 to be considered are:

- ANA testing is not helpful in confirming a diagnosis of rheumatoid arthritis or osteoarthritis therefore should not be used in such conditions.

- ANA testing is not recommended to evaluate fatigue, back pain or other musculoskeletal pain unless accompanied by one or more of the clinical features in favor of a CTD.

- ANA testing should usually be ordered only once.

- Positive ANA tests do not need to be repeated.

- Negative tests need to be repeated only if there is a strong suspicion of an evolving CTD or a change in the patient's illness suggesting the diagnosis should be revised.

- A positive ANA test is important only in conjunction with clinical evaluation and in the absence of symptoms and signs of a CTD; a positive ANA test only confounds the diagnosis. A positive ANA test can also be seen in healthy individuals, particularly the elderly or in a wide range of diseases other than CTD, where it has no diagnostic or prognostic value.

Recommendations in the guidelines may further evolve over time, as newer analytic methods and additional clinical research yield important results.

The future for ANA detection looks very promising. We have come a long way from the simplest test like LE cell method to fully automated ELISAs to nanotechnology. Future development will undoubtedly include more sophisticated instrumentation with ultra sensitive detection, faster turnaround time, and increased throughput in ANA detection. Advances in the new technologies like multiplex immunoassays and antigen microarrays offer an attractive alternative to traditional ELISA, immunoblot, and IFA techniques. Rapid development in the area of quantum dots and other fluorescent nanoparticles will also eventually benefit routine clinical laboratory analysis.

The authors declare that they have no competing interests.

YK is primarily responsible for design of the study, literature search and drafting of the manuscript, AB and RWM participated in the sequence alignment and made critical revision for important intellectual content. All authors read and approved the final manuscript.