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Hep-2000 Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Hep-2000 and links to relevant pages within the course.

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Antinuclear Antibody Testing: Methods and Pattern Interpretation
Cell Morphology

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.

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History of ANA Testing

Slide-based ANA testing using a cell substrate started in the 1950s and continues to be the gold standard method. In the early days of ANA testing, rodent tissue (stomach, liver and/or kidney) was commonly used as the substrate. Rodent tissue however had several drawbacks such as small cell size, a lack of dividing cells (mitotics) and poor antigen expression that made interpretation of ANA patterns difficult. In the 1980s, cultured cell lines were examined for utility as an ANA substrate and the human epithelial- like cell line HEp-2 gained popularity. HEp-2's advantages over rodent tissue are: A large nucleus Better antigen expression Abundant mitotic cells that assist in interpretation of the ANA pattern (if grown properly).More recently a cell line called HEp-2000® has become popular for ANA detection. HEp-2000® is a HEp-2 cell line that has been transfected with the cDNA for overexpression of the SSA/Ro antigen. This results in a substrate with all of the original advantages of HEp-2 plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen and the ability to identify these clinically significant antibodies during the screening process.(Ref4)It has also been demonstrated that antibodies to SSA/Ro develop early in the disease process.(Ref5) Perhaps most importantly, if a woman has anti-SSA/Ro antibodies and becomes pregnant there is a risk of the antibodies crossing the placenta, resulting in the fetus developing neonatal lupus and congenital heart block in utero.The advantage of using these transfected cells is documented in the current Clinical and Laboratory Standards Institute (CLSI) guidelines for ANA testing. Here they note the "dramatically increased" sensitivity of transfected cells for the detection of SS-A/Ro and the unaltered effect of transfection on other ANA patterns.(Ref6)

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Reading ANA Patterns Using a HEp-2 or HEp-2000® Substrate

The tendency in interpretation of ANA results (reading the slides) is to over-read and call negative samples positive. Most frequently this is caused by staring too long at the cells, using too high of a magnification and an understandable desire to detect all true positives. With proper training reading the slides can be greatly simplified. Follow these three steps:Look initially at 200x total magnification.An ANA positive sample must have a clearly discernable pattern in the nucleus of the interphase cells.Only use 400x magnification to confirm the pattern seen during initial the screen with 200x.It is advised that the ANA slide first be viewed using 200x total magnification (20x objective with 10x oculars). During this first assessment you look for a clearly discernable pattern in the nucleus of the interphase cells. If no discernable pattern is seen the sample is ANA negative.Do not stare so long you start to hallucinate and see patterns that are not there. Do not switch to a higher magnification just to see if you can see something that isn't visible at 200x. If the sample looks negative at 200x and you go to 400x and start thinking "I might be able to somewhat see a pattern" the sample is negative. It cannot be overemphasized that to be ANA positive there must be a clearly discernable pattern in the nucleus of the interphase cells.

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Antigen Chart

Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA

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SSA/Ro Pattern

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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Homogeneous and SSA/Ro (on HEp-2000®)

One of the advantages to using the HEp-2000® substrate is the ability to identify the presence of anti-SSA/Ro antibodies during the ANA screen.In this sample we see the homogeneous ANA pattern, smooth staining in the interphase cells and smooth staining of the chromosomal area of the mitotics (a) along with the hyperexpressing cells demonstrating the SSA/Ro pattern (b).In cases of mixed homogeneous and SSA/Ro, follow-up testing for anti-dsDNA and anti-ENA antibodies is necessary.When mixed patterns are titered, the endpoint for each pattern is reported.This pattern is reported as ANA positive, Homogeneous and SSA/Ro pattern, anti-SSA/Ro antibodies present; titering is necessary.Homogeneous and SSA/Ro:Interphase cellsSmooth staining of the entire nucleus (homogeneous)Hyperexpressing SS-A/Ro pattern in 10-15%Metaphase mitotic cellsSmooth staining of the chromosome regionRegion outside of chromosome area may stain

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Other Mixed Patterns

Mixed patterns are not limited just to the ones viewed previously. Virtually any combination is possible.Other Combinations: Centromere and SS-A/Ro (HEp-2000®) Homogeneous, speckled, and SS-A/Ro (HEp-2000®) Homogeneous and nucleolar Both patterns may be caused by antibodies to one antigen Remember: any combination is possible!

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