complex in the nucleolus. there are ten proteins in this complex and antibodies to eight of them are found at varying frequencies; pm/scl-100 (70–80%), pm/scl-75 (46–80%), hrrp4 (50%), hrrp42 (21%), hrrp46 (18%), hcs14 (14%), hrrp41 (10%) and hrrp40 (7%). anti-dfs70 antibodies anti-dfs70 antibodies generate a dense fine speckled pattern in indirect immunofluorescence and are found in normals and in various conditions, but are not associated with a systemic autoimmune pathology. therefore, they can be used to help to rule out such conditions in ana positive individuals. a significant number of patients are diagnosed as systemic lupus erythematosus or undifferentiated connective tissue disease largely based on a positive ana. in case no defined autoantibody can be detected (e.g. anti-ena antibodies), the testing of anti-dfs70 antibodies is recommended to verify the diagnosis. anti-dfs70 antibody tests are available as ce-marked tests. until now, no fda cleared assay is available. ana test kit for carrying out a test for antinuclear antibodies stages of immunofluorescence for the detection of antinuclear antibodies. hep-2 cells are permeablised (1) and then incubated with a person's blood serum (2). if the serum contains antibodies, they will bind to antigens within the hep-2 cell nucleus. these antibodies can be visualised by subsequent incubation with anti-human antibodies conjugated to a fluorescent molecule (3). the presence of anas in blood can be confirmed by a screening test. although there are many tests for the detection of anas, the most common tests used for screening are indirect immunofluorescence and enzyme-linked immunosorbent assay (elisa). following detection of anas, various subtypes are determined. indirect immunofluorescence indirect immunofluorescence is one of the most commonly used tests for anas. typically, hep-2 cells are used as a substrate to detect the antibodies in human serum. microscope slides are coated with hep-2 cells and the serum is incubated with the cells. if the said and targeted antibodies are present then they will bind to the antigens on the cells; in the case of anas, the antibodies will bind to the nucleus. these can be visualised by adding a fluorescent tagged (usually fitc or rhodopsin b) anti-human antibody that binds to the antibodies. the molecule will fluoresce when a specific wavelength of light shines on it, which can be seen under the microscope. depending on the antibody present in the human serum and the localisation of the antigen in the cell, distinct patterns of fluorescence will be seen on the hep-2 cells. levels of antibodies are analysed by performing dilutions on blood serum. an ana test is considered positive if fluorescence is seen at a titre of 1:40/1:80. higher titres are more clinically significant as low positives (≤1:160) are found in up to 20% of healthy individuals, especially the elderly. only around 5% of the healthy population have ana titres of 1:160 or higher. hep-2 nucleolar staining pattern of anas. until around 1975, when hep-2 cells were introduced, animal tissue was used as the standard substrate for immunofluorescence. hep-2 cells are currently one of the most common substrates for ana detection by immunofluorescence. originally started a laryngeal carcinoma strain, the cell line was contaminated and displaced by hela cells, and has now been identified as actually hela cells. they are superior to the previously used animal tissues because of their large size and the high rate of mitosis (cell division) in the cell line. this allows the detection of antibodies to mitosis-specific antigens, such as centromere antibodies. they also allow identification of anti-ro antibodies, because acetone is used for fixation of the cells (other fixatives can wash the antigen away). there are many nuclear staining patterns seen on hep-2 cells: homogeneous, speckled, nucleolar, nuclear membranous, centromeric, nuclear dot and pleomorphic. the homogeneous pattern is seen when the condensed chromosomes and interphase chromatin stain. this pattern is associated with anti-dsdna antibodies, antibodies to nucleosomal components, and anti-histone antibodies. there are two speckled patterns: fine and coarse. the fine speckled pattern has fine nuclear staining with unstained metaphase chromatin, which is associated with anti-ro and anti-la antibodies. the coarse staining pattern has coarse granular nuclear staining, caused by anti-u1-rnp and anti-sm antibodies. the nucleolar staining pattern is associated with many antibodies including anti-scl-70, anti-pm-scl, anti-fibrillarin and anti-th/to. nuclear membrane staining appears as a fluorescent ring around the cell nucleus and are produced by anti-gp210 and anti-p62 antibodies. the centromere pattern shows multiple nuclear dots in interphase and mitotic cells, corresponding to the number of chromosomes in the cell. nuclear dot patterns show between 13 and 25 nuclear dots in interphase cells and are produced by anti-sp100 antibodies. pleomorphic pattern is caused by antibodies to the proliferating cell nuclear antigen. indirect immunofluorescence has been shown to be slightly superior compared to elisa in detection of ana from hep-2 cells. crithidia luciliae immunofluorescence staining pattern of anti-dsdna antibodies on c. luciliaesubstrate. the kinetoplast located near the flagellum is stained, indicating the presence of anti-dsdna antibodies in a person with systemic lupus erythamatosus. crithidia luciliae are haemoflaggelate single celled protists. they are used as a substrate in immunofluorescence for the detection of anti-dsdna antibodies. they possess an organelle known as the kinetoplast which is a large mitochondrion with a network of interlocking circular dsdna molecules. after incubation with serum containing anti-dsdna antibodies and fluorescent-labelled anti-human antibodies, the kinetoplast will fluoresce. the lack of other nuclear antigens in this organelle means that using c. luciliae as a substrate allows for the specific detection of anti-dsdna antibodies. elisa enzyme-linked immunosorbent assay (elisa) uses antigen-coated microtitre plates for the detection of anas. each well of a microtitre plate is coated with either a single antigen or multiple antigens to detect specific antibodies or to screen for anas, respectively. the antigens are either from cell extracts or recombinant. blood serum is incubated in the wells of the plate and is washed out. if antibodies that bind to antigen are present then they will remain after washing. a secondary anti-human antibody conjugated to an enzyme such as horseradish peroxidase is added. the enzyme reaction will produce a change in colour of the solution that is proportional to the amount of antibody bound to the antigen. there are significant differences in the detection of ana by immunofluorescence and different elisa kits and there is only a marginal agreement between these. a clinician must be familiar with the differences in order to evaluate the outcomes of the various assays. sensitivity the following table lists the sensitivity of different types of anas for different diseases. ana typetarget antigensensitivity (%) sledrug-induced lediffuse systemic sclerosislimited systemic sclerodermasjögren syndromeinflammatory myopathymctd all anas (by indirect if) various 9510080807040–6095 anti-dsdna dna 60–––30–- anti-sm core proteins of snrnps 40–––––- anti-histone histones 6090––––- anti scl-70 type i topoisomerase ––2010––- anti-centromere centromeric proteins ––3080––- ss-a (ro) rnps 40–––5010- ss-b (la) rnps 10–15–––60–90– – = less than 5% sensitivity some anas appear in several types of disease, resulting in lower specificity of the test. for example, igm-rheumatoid factor (igm-rf) have been shown to cross-react with ana giving falsely positive immunofluorescence. positive ana as well as anti-dna antibodies have been reported in patients with autoimmune thyroid disease. ana can have a positive test result in up to 45% of people with autoimmune thyroid conditions or rheumatoid arthritis and up to 15% of people with hiv or hepatitis c. as per lupus foundation of america, "about 5% of the general population will have a positive ana. however, at least 95% of the people who have a positive ana do not have lupus. a positive ana test can sometimes run in families, even if family members have no evidence of lupus." on the other hand, they say, although 95% of the patients who actually have lupus test positive for ana, "only a small percentage have a negative ana, and many of those have other antibodies (such as anti-phospholipid antibodies, anti-ro, anti-ssa) or their ana converted from positive to negative from steroids, cytotoxic medications, or uremia (kidney failure)." history le cell the le cell was discovered in bone marrow in 1948 by hargraves et al. in 1957 holborow et al. first demonstrated ana using indirect immunofluorescence. this was the first indication that processes affecting the cell nucleus were responsible for sle. in 1959 it was discovered that serum from individuals with sle contained antibodies that precipitated with saline extracts of nuclei, known as extractable nuclear antigens (enas). this led to the characterisation of ena antigens and their respective antibodies. thus, anti-sm and anti-rnp antibodies were discovered in 1966 and 1971, respectively. in the 1970s, the anti-ro/anti-ss-a and anti-la/anti-ss-b antibodies were discovered. the scl-70 antibody was known to be a specific antibody to scleroderma in 1979, however the antigen (topoisomerase-i) was not characterised until 1986. the jo-1 antigen and antibody were characterised in 1980.