antinuclear antibody antinuclear antibody antinuclear antibodies (anas, also known as antinuclear factor or anf) are autoantibodies that bind to contents of the cell nucleus. in normal individuals, the immune system produces antibodies to foreign proteins (antigens) but not to human proteins (autoantigens). in some cases, antibodies to human antigens are produced. homogeneous immunofluorescence staining pattern of double stranded dna antibodies on hep-20-10 cells. interphase cells show homogeneous nuclear staining while mitotic cells show staining of the condensed chromosome regions. there are many subtypes of anas such as anti-ro antibodies, anti-la antibodies, anti-sm antibodies, anti-nrnp antibodies, anti-scl-70 antibodies, anti-dsdna antibodies, anti-histone antibodies, antibodies to nuclear pore complexes, anti-centromere antibodies and anti-sp100 antibodies. each of these antibody subtypes binds to different proteins or protein complexes within the nucleus. they are found in many disorders including autoimmunity, cancer and infection, with different prevalences of antibodies depending on the condition. this allows the use of anas in the diagnosis of some autoimmune disorders, including systemic lupus erythematosus, sjögren syndrome, scleroderma, mixed connective tissue disease, polymyositis, dermatomyositis, autoimmune hepatitis and drug induced lupus. the ana test detects the autoantibodies present in an individual's blood serum. the common tests used for detecting and quantifying anas are indirect immunofluorescence and enzyme-linked immunosorbent assay (elisa). in immunofluorescence, the level of autoantibodies is reported as a titre. this is the highest dilution of the serum at which autoantibodies are still detectable. positive autoantibody titres at a dilution equal to or greater than 1:160 are usually considered as clinically significant. positive titres of less than 1:160 are present in up to 20% of the healthy population, especially the elderly. although positive titres of 1:160 or higher are strongly associated with autoimmune disorders, they are also found in 5% of healthy individuals. autoantibody screening is useful in the diagnosis of autoimmune disorders and monitoring levels helps to predict the progression of disease. a positive ana test is seldom useful if other clinical or laboratory data supporting a diagnosis are not present. immunity and autoimmunity the human body has many defense mechanisms against pathogens, one of which is humoral immunity. this defence mechanism produces antibodies (large glycoproteins) in response to an immune stimulus. many cells of the immune system are required for this process, including lymphocytes (t-cells and b-cells) and antigen presenting cells. these cells coordinate an immune response upon the detection of foreign proteins (antigens), producing antibodies that bind to these antigens. in normal physiology, lymphocytes that recognise human proteins (autoantigens) either undergo programmed cell death (apoptosis) or become non-functional. this self-tolerance means that lymphocytes should not incite an immune response against human cellular antigens. sometimes, however, this process malfunctions and antibodies are produced against human antigens, which may lead to autoimmune disease. ana subtypes anas are found in many disorders, as well as some healthy individuals. these disorders include: systemic lupus erythematosus (sle), rheumatoid arthritis, sjögren syndrome, scleroderma, polymyositis, dermatomyositis, primary biliary cirrhosis, drug induced lupus, autoimmune hepatitis, multiple sclerosis, discoid lupus, thyroid disease, antiphospholipid syndrome, juvenile idiopathic arthritis, psoriatic arthritis, juvenile dermatomyositis, idiopathic thrombocytopaenic purpura, infection and cancer. these antibodies can be subdivided according to their specificity, and each subset has different propensities for specific disorders. extractable nuclear antigens extractable nuclear antigens (ena) are a group of autoantigens that were originally identified as antibody targets in people with autoimmune disorders. they are termed ena because they can be extracted from the cell nucleus with saline. the enas consist of ribonucleoproteins and non-histone proteins, named by either the name of the donor who provided the prototype serum (sm, ro, la, jo), or the name of the disease setting in which the antibodies were found (ss-a, ss-b, scl-70). anti-ro/ss-a and anti-la/ss-b speckled immunofluorescence staining pattern of anti-nuclear antibodies on hep-20-10 cells. this staining pattern is seen with anti-ro and anti-la antibodies. anti-ro and anti-la antibodies, also known as ss-a and ss-b, respectively, are commonly found in primary sjögren's syndrome, an autoimmune disorder that affects the exocrine glands. the presence of both antibodies is found in 30–60% of sjögren's syndrome, anti-ro antibodies alone are found in 50–70% of sjögren's syndrome and 30% of sle with cutaneous involvement, and anti-la antibodies are rarely found in isolation. anti-la antibodies are also found in sle; however, sjögren's syndrome is normally also present. anti-ro antibodies are also found less frequently in other disorders including autoimmune liver diseases, coeliac disease, autoimmune rheumatic diseases, cardiac neonatal lupus erythematosus and polymyositis. during pregnancy, anti-ro antibodies can cross the placenta and cause heart block and neonatal lupus in babies. in sjögren's syndrome, anti-ro and anti-la antibodies correlate with early onset, increased disease duration, parotid gland enlargement, disease outside the glands and infiltration of glands by lymphocytes. anti-ro antibodies are specific to components of the ro-rnp complex, comprising 45kda, 52kda, 54kda and 60kda proteins and rna. the 60kda dna/rna binding protein and 52kda t-cell regulatory protein are the best characterised antigens of anti-ro antibodies. collectively, these proteins are part of a ribonucleoprotein (rnp) complex that associate with the human y rnas, hy1-hy5. the la antigen is a 48kda transcription termination factor of rna polymerase iii, which associates with the ro-rnp complex. the mechanism of antibody production in sjögren's syndrome is not fully understood, but apoptosis (programmed cell death) and molecular mimicry may play a role. the ro and la antigens are expressed on the surface of cells undergoing apoptosis and may cause the inflammation within the salivary gland by interaction with cells of the immune system. the antibodies may also be produced through molecular mimicry, where cross reactive antibodies bind to both virus and human proteins. this may occur with one of the antigens, ro or la, and may subsequently produce antibodies to other proteins through a process known as epitope spreading. the retroviral gag protein shows similarity to the la protein and is proposed as a possible example for molecular mimicry in sjögren's syndrome. anti-sm anti-smith (anti-sm) antibodies are a very specific marker for sle. approximately 99% of individuals without sle lack anti-sm antibodies, but only 20% of people with sle have the antibodies. they are associated with central nervous system involvement, kidney disease, lung fibrosis and pericarditis in sle, but they are not associated with disease activity. the antigens of the anti-sm antibodies are the core units of the small nuclear ribonucleoproteins (snrnps), termed a to g, and will bind to the u1, u2, u4, u5 and u6 snrnps. most commonly, the antibodies are specific for the b, b' and d units. molecular and epidemiological studies suggest that anti-sm antibodies may be induced by molecular mimicry because the protein shows some similarity to epstein-barr virus proteins. anti-nrnp/anti-u1-rnp anti-nuclear ribonucleoprotein (anti-nrnp) antibodies, also known as anti-u1-rnp antibodies, are found in 30–40% of sle. they are often found with anti-sm antibodies, but they may be associated with different clinical associations. in addition to sle, these antibodies are highly associated with mixed connective tissue disease. anti-nrnp antibodies recognise the a and c core units of the snrnps and because of this they primarily bind to the u1-snrnp. the immune response to rnp may be caused by the presentation of the nuclear components on the cell membrane in apoptotic blebs. molecular mimicry has also been suggested as a possible mechanism for the production of antibodies to these proteins because of similarity between u1-rnp polypeptides and epstein-barr virus polypeptides. anti-scl-70/anti-topoisomerase i anti-scl-70 antibodies are linked to scleroderma. the sensitivity of the antibodies for scleroderma is approximately 34%, but is higher for cases with diffuse cutaneous involvement (40%), and lower for limited cutaneous involvement (10%). the specificity of the antibodies is 98% and 99.6% in other rheumatic diseases and normal individuals, respectively. in addition to scleroderma, these antibodies are found in approximately 5% of individuals with sle. the antigenic target of anti-scl-70 antibodies is topoisomerase i. anti-jo-1 although anti-jo-1 antibodies are often included with anas, they are actually antibodies to the cytoplasmic protein, histidyl-trna synthetase - an aminoacyl-trna synthetase essential for the synthesis of histidine loaded trna. they are highly associated with polymyositis and dermatomyositis, and are rarely found in other connective tissue diseases. around 20–40% of polymyositis is positive for jo-1 antibodies and most will have interstitial lung disease, hla-dr3 and hla-drw52 human leukocyte antigen (hla) markers; collectively known as jo-1 syndrome. anti-dsdna dsdna antibody. the variable regions (yellow) are complementary to the dsdna strands. these antibodies are found commonly in the sera of people with sle. anti-double stranded dna (anti-dsdna) antibodies are highly associated with sle. they are a very specific marker for the disease, with some studies quoting nearly 100%. data on sensitivity ranges from 25 to 85%. anti-dsdna antibody levels, known as titres, correlate with disease activity in sle; high levels indicate more active lupus. the presence of anti-dsdna antibodies is also linked with lupus nephritis and there is evidence they are the cause. some anti-dsdna antibodies are cross reactive with other antigens found on the glomerular basement membrane (gbm) of the kidney, such as heparan sulphate, collagen iv, fibronectin and laminin. binding to these antigens within the kidney could cause inflammation and complement fixation, resulting in kidney damage. presence of high dna-binding and low c3 levels have been shown to have extremely high predictive value (94%) for the diagnosis of sle. it is also possible that the anti-dsdna antibodies are internalised by cells when they bind membrane antigens and then are displayed on the cell surface. this could promote inflammatory responses by t-cells within the kidney. it is important to note that not all anti-dsdna antibodies are associated with lupus nephritis and that other factors can cause this symptom in their absence. the antigen of anti-dsdna antibodies is double stranded dna. anti-histone antibodies anti-histone antibodies are found in the serum of up to 75–95% of people with drug induced lupus and 75% of idiopathic sle. unlike anti-dsdna antibodies in sle, these antibodies do not fix complement. although they are most commonly found in drug induced lupus, they are also found in some cases of sle, scleroderma, rheumatoid arthritis and undifferentiated connective tissue disease. many drugs are known to cause drug induced lupus and they produce various antigenic targets within the nucleosome that are often cross reactive with several histone proteins and dna. procainamide causes a form of drug-induced lupus that produces antibodies to the histone h2a and h2b complex. anti-gp210 and anti-p62 both anti-glycoprotein-210 (anti-gp210) and anti-nucleoporin 62 (anti-p62) antibodies are antibodies to components of the nuclear membrane and are found in primary biliary cirrhosis (pbc). each antibody is present in approximately 25–30% of pbc. the antigens of both antibodies are constituents of the nuclear membrane. gp210 is a 200kda protein involved in anchoring components of the nuclear pore to the nuclear membrane. the p62 antigen is a 60kda nuclear pore complex. anti-centromere antibodies immunofluorescence staining pattern of anti-centromere antibodies on hep-20-10 cells. anti-centromere antibodies are associated with limited cutaneous systemic sclerosis, also known as crest syndrome, primary biliary cirrhosis and proximal scleroderma. there are six known antigens, which are all associated with the centromere; cenp-a to cenp-f. cenp-a is a 17kda histone h3-like protein. cenp-b is an 80kda dna binding protein involved in the folding of heterochromatin. cenp-c is a 140kda protein involved in kinetochore assembly. cenp-d is a 50kda protein of unknown function, but may be homologous to another protein involved in chromatin condensation, rcc1. cenp-e is a 312kda protein from the kinesin motor protein family. cenp-f is a 367kda protein from the nuclear matrix that associates with the kinetochore in late g2 phase during mitosis. cenp-a, b and c antibodies are most commonly found (16–42% of systemic sclerosis) and are associated with raynaud's phenomenon, telangiectasias, lung involvement and early onset in systemic sclerosis. anti-sp100 anti-sp100 antibodies are found in approximately 20–30% of primary biliary cirrhosis (pbc). they are found in few individuals without pbc, and therefore are a very specific marker of the disease. the sp100 antigen is found within nuclear bodies; large protein complexes in the nucleus that may have a role in cell growth and differentiation. anti-pm-scl anti-pm-scl antibodies are found in up to 50% of polymyositis/systemic sclerosis (pm/ssc) overlap syndrome. around 80% of individuals with antibodies present in their blood serum will have the disorder. the presence of the antibodies is linked to limited cutaneous involvement of pm/ssc overlap syndrome. the antigenic targets of the antibodies are components of the rna-processing exosome