This slide is an illustration of a HEp-2 or HEp-2000® cell with several nuclear and cytoplasmic structures indicated. Antibodies to DNA, histones, centromere, nuclear RNP, cytoplasmic RNP, mitochondria, ribosomes, lysosomes, golgi apparatus, as well as a variety of cytoskeletal proteins such as microfilaments, intermediate filaments, and microtubules, can be detected using the HEp-2 or HEp-2000® cell lines.(Ref10) It is important to note that RNA constitutes the bulk of the nucleic acid in the cells, being 5-10 times more abundant than DNA.(Ref11) Generally we consider 2 compartments in the cell when detecting autoantibodies in systemic rheumatic disease: 1) nucleus, and 2) cytoplasm. Staining of the nucleus includes staining inside the nucleoli.
SSA/Ro on HEp-2000®This is an example of the SSA/Ro ANA pattern using the HEp-2000® substrate. (This hyperexpressing pattern is not seen on standard HEp-2 substrates.)As stated earlier, the HEp-2000® substrate utilizes genetically engineered HEp-2 cells with increased expression of SS-A/Ro antigen in the cells.When the patient sample contains autoantibodies to the SS-A/Ro antigen, approximately 90% of the time the sample will produce a distinctive pattern with 10 - 15% of the hyperexpressing cells showing strong speckled staining, frequently with strong nucleolar staining (a). The metaphase mitotic cells are negative (b). The remaining cells demonstrate weak speckled and nucleolar staining (c). The presence of anti-SSA/Ro antibodies is confirmed by the unique staining pattern. Follow-up testing for antibodies to other extractable nuclear antigens (ENAs) is recommended. These antibodies are seen in patients with SLE, Sjögren's syndrome and low frequency in other diseases.This pattern is reported as ANA positive, SSA/Ro pattern, anti-SSA/Ro antibodies present. Most labs will titer these samples. However, because the significance is that the anti-SSA antibodies are present regardless of the titer endpoint, some labs do not titer the SSA/Ro pattern.SS-A/Ro (HEp-2000® only) Interphase:Seen with about 89% of samples containing anti-SS-A/Ro antibodiesHyperexpressing interphase cells 10-15% hyperexpress the SS-A/Ro antigen Stronger nucleolar staining Stronger speckled staining Non-hyperexpressing interphase cells May or may not stainSS-A/Ro (HEp-2000® only) Metaphase:Metaphase mitotic cells No staining in the chromosome region Region outside of the chromosome area will stain with variable intensitySome mitotics may also demonstrate brighter staining
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