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Antinuclear antibodies: clinical applications. AA WanchuDepartment of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012, India., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 0011013488
One of the common serological hallmarks of autoimmune disorders is the presence of various autoantibodies in the sera of patients affected by these disorders. Antinuclear antibodies (ANA) detection is often needed to aid the diagnosis in several autoimmune disorders. In view of the different methodologies available for their detection, it becomes essential to understand the advantages and pitfalls of each procedure. This brief review discusses some methodological aspects of ANA detection and the clinical relevance of the presence of some of the autoantibodies found in the sera of patients with autoimmune disorders. Keywords: Antibodies, Antinuclear, blood,Autoimmune Diseases, diagnosis,Fluorescent Antibody Technique, Indirect, Human, Lupus Erythematosus, Systemic, diagnosis,
Antinuclear antibodies (ANA) are a diverse group of auto-antibodies that are found in a host of systemic autoimmune disorders that include systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS), systemic sclerosis (SSc), inflammatory myositis (IM), mixed connective tissue disorder (MCTD) and rheumatoid arthritis (RA). The objective of this brief review is to discuss some aspects of ANA testing and its relevance to the practicing clinician.
Historically, in 1948 the first formal description of ANA related phenomenon was made in which concentrated bone marrow of patients with SLE was found to contain LE cells[1]. These were later used as supportive evidence for diagnosing SLE, SS and RA. The LE cell was soon found to be due to an antibody directed against deoxyribonucleoprotein that worked as an opsonin to phagocytose antibody sensitised nuclei by polymorphonuclear neutrophils. In the late 1950s, a novel technique that utilised indirect immunofluorescence (IF) was developed[2]. This was found to provide a more sensitive assay for the detection of the autoimmune phenomenon. Subsequent studies further characterised the Sm antigen-antibody system among patients with SLE and the nuclear ribonucleoprotein (nRNP) antigen-antibody system in patients with MCTD[3]. Presently, a large number of antigens have been found and in some cases they have been assigned a role in the pathophysiology of the disease[4]. The prevalence of ANA in various disorders is shown in [Table - 1][5].
Indirect immunofluorescence: IF has become a simple, reliable and highly sensitive tool for screening purposes in the detection of ANA. Briefly, patient’s sera are used in different dilutions and incubated with substrate cells. Ideally, cells from the human cell lines like those from the human epithelial cell line, HEp-2, should be used. However, when in settings where there may be financial constraints for maintaining cell lines, the use of rodent e.g. rat liver sections can be a substitute with some limitations. Bound antibodies are tagged by fluorescein-conjugated anti-human immunoglobulin and seen through a fluorescent microscope. This is the currently preferred method for testing for ANA. As the target antigens vary among different ANAs, the fluorescence pattern can provide clinically useful information [6]. These patterns may be homogeneous, rim or speckled if they are nuclear. Alternatively, they might have a nucleolar pattern in which case the patterns may be discrete speckled, grainy speckled and clumpy staining. [Table:II] shows various staining patterns along with their disease associations. Immunodiffusion: This method uses Ouchterlony technique for double-diffusion in an agarose gel in which calf or rabbit tissue extract that contains extractable nuclear antigens are placed in wells adjacent to patients serum. In 24 to 48 hours, the antigens and antibodies diffuse towards each other forming precipitin lines. The antibodies to the following antigens can be detected by this technique: snRNP, Ro, La, Ku, Topoisomerase I, PM/Scl. The major drawbacks of this technique is that it is insensitive in comparison with other techniques, needs larger quantities of immunoglobulins to form precipitate lines and does not detect some unstable antigens. Counter immunoelectrophoresis: This is a modification of the previous technique that needs lesser antibody and can detect acidic antigens. The latter limits its utility and this technique have been largely replaced other methods. Enzyme-Linked Immunosorbent Assay (ELISA): This is a highly sensitive and fast method for ANA detection. Several clinical laboratories use commercial kits for detecting specific auto-antibodies. Sometimes false-positives results are produced and further tests may be needed for confirmation. Miscellaneous techniques: Other techniques that are in use include immunoprecipitation, Western blotting and radioimmunoassay (RIA). Of these Western blotting are in use largely for research purposes. RIA is used for detecting anti-dsDNA antibodies (Farr RIA). A positive ANA test in itself does not make the diagnosis of any disorder. The test has to be interpreted in the relevant clinical context. Other than in rare situation where an error in testing has been suspected, immediate repetition of the test is not necessary. Since the clinical progression of the disease can be progressive and may evolve over a period of time, it might be useful to repeat the test at a subsequent point of time. This is especially true if the clinical course of the disease changes in that time period. If Hep-2 cells are used as substrate then the chances of false-negative ANA are considerably minimised. Disorders commonly associated with antinuclear antibodies are discussed below.
Anti-DNA antibodies: These include anti ssDNA-antibodies that target purine and pyrimidine bases of denatured DNA and dsDNA antibodies that target the ribose phosphate backbone of native DNA. Although several disorders are associated with anti-ssDNA antibodies, the presence of anti-dsDNA antibodies in higher titers are relatively specific for SLE. These appear in approximately 73% patients at some time or the other during the disease course[7]. Low levels of these antibodies are sometimes seen in normal persons, and individuals with SS and RA. In SLE their presence correlates with the presence of nephritis and disease activity. They might contribute to disease pathology, too. Anti-sn RNP antibodies: Antibodies against small nuclear ribonucleoproteins (sn-RNP) are directed towards the RNAs or proteins involving splicing pre-mRNA. One group includes U1, U2, U4/U6, U7, U11 and U12 snRNPs where U represents uridine-rich RNA [8]. These are associated with disease activity, myositis, oesophageal hypomotility, Raynaud’s phenomenon, infrequent nephritis, arthralgias, arthritis, sclerodactyly and interstitial pneumonitis. The other group of proteins are anti-Sm antibodies that target the Sm protein. These are relatively specific and their presence is associated with milder renal and central nervous system disease and disease flares. Anti-histone antibodies: These are directed against the protein components of nucleosomes and include several proteins that have been labelled as H1, H2A, H2B, H3, and H4[9]. These are present in all cases of drug induced lupus, but may also appear in a smaller percentage of cases of RA, primary biliary cirrhosis, scleroderma, Epstein-Barr virus infection and some malignancies. Anti-Ro (SSA) and anti-La (SSB) antibodies: These two antibodies target two different ribonucleoprotein particles. The former is associated with photosensitivity, lung disease, lymphopaenia and in some cases nephritis. The latter are associated with late-onset SLE, secondary SS, neonatal lupus erythematosus and protection from anti-Ro associated nephritis[10].
Anti-centromere (kinetochore) antibodies: These are directed against components of the mitotic spindle apparatus. Three centromere (CENP) proteins have been identified as target antigens: CENP-A, CENP-C and CENP-D. Their presence correlates with Raynaud’s phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasias and limited cutaneous involvement. In some cases of diffuse cutaneous disease, Hashimoto’s thyroiditis and primary biliary cirrhosis there may be a variable incidence. Anti-topoisomerase I (Scl-70): These are directed against a component of DNA topoisomerase I enzyme. These antibodies are associated with generalized skin involvement among patients with SSc, pulmonary fibrosis, cardiac involvement and digital pitting scars. Along with anti-centromere antibodies these are important in classifying patients with SSc as limited and diffuse subtypes.
Anti-Ro antibodies: The prevalence of these autoantibodies varies between 40 to 95%. These are associated with the presence of extra-glandular manifestations that include vasculitis, neurological involvement, glandular dysfunction, low haemoglobin, total leukocyte and platelet counts, and rheumatoid factor. Anti-La antibodies: These may be present in the serum of up to 87% patients with SS. Their presence is associated with manifestations similar to anti-Ro antibodies, namely, extra-glandular manifestations that include vasculitis, neurological involvement, glandular dysfunction, low haemoglobin, total leukocyte and platelet counts and rheumatoid arthritis.
Anti-synthetase antibodies: These are highly antigen- and disease- specific and include five different aminoacyl-transfer RNA synthetases namely, anti-Jo1, PL-7, PL-12, EJ and OJ. These targets the synthetases for histidine, threonine, alanine, glycine and isoleucine, respectively. Of these, anti-Jo-1 antibodies are present in 30% patients with inflammatory myositis and appear more commonly among patients with polymyositis. Together these antibodies characterise the “anti-synthetase syndrome” which includes interstitial lung disease, arthritis, Raynaud’s phenomenon, sclerodactyly, facial telangiectasia, calcinosis and Sicca syndrome. Anti-SRP (signal recognition particle) antibodies: These are directed against cytoplasmic proteins that facilitate translocation of various proteins across the endoplasmic reticulum. These are seen in 4% of individuals with myositis and are associated with acute onset, severe disease, and relative resistance to therapy and higher mortality [12]. Anti-Mi-2 antibodies: These occur predominantly among patients with dermatomyositis and are associated with the so-called “V” and “shawl” signs. Anti-PM-Scl antibodies: These are seen in 50% patients with overlap with scleroderma and are associated with the presence of arthritis, dermatomyositis, skin lesions, calcinosis, mechanic’s hands and eczema. Anti-snRNP antibodies: These are predominantly anti-U1 snRNP antibodies and are generally associated with the presence of mixed connective tissue disease, SLE-myositis overlap, myositis-scleroderma overlap and undifferentiated connective tissue disease.
ANA’s are also present in lower titers in several other disorders that include liver diseases, leprosy, multiple sclerosis, silicone gel implants, Henoch Schonlein purpura, juvenile rheumatoid arthritis, myasthenia gravis, etc. The clinical significance of these antibodies in these settings remains unclear. In general, however, the presence of ANA’s in the serum would warrant a follow up with more specific assays. Their presence remains adjunctive rather than diagnostic for any particular disorder. The role of some of the more recently described antibodies remains to be clarified [14]. Results of ANA testing have to be analysed in the clinical context and in most situations the patients and not the serology test needs to be treated.
[Table - 1], [Table - 2], [Table - 3]
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