Hep-2 Information and Courses from MediaLab, Inc.

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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.	View Page
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)	View Page
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.	View Page
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	View Page
References American College of Rheumatology, Committee on Rheumatologic Care, Position Statement, Methodology of Testing for Antinuclear Antibodies; Feb, 2009. Available at http://www.rheumatology.org/search/search.asp accessed on June 16, 2010Anuradah V, Chopra A, Sturgess A, Edmonds J. Cost-effective screening method for antinuclear antibody detection. Asian Pacific League of Associations for Rheumatology. 2004(7):13-18.Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct 16 2003;349(16):1526-1533.Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem. 10 7 2004;50(12):2361-2369.Clinical and Laboratory Standards Institute (formerly NCCLS); Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved Guidelines - Second Edition. CLSI I/LA2-A2. 2006;26(13).Fritzler MJ, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal. 2002;16:103-108.Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J.Clin.Lab.Anal. 1995;9:218-224.Fritzler MJ, Wall W, Gohill J, Kinsella TD, Humbel RL. The Detection of Autoantibodies on HEp-2 Cells Using an Indirect Immunoperoxidase Kit (Colorzyme®). Diag Immunol. 1986;4:217-221. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology. 1996;28:54-57.Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells. Clin.Immunol.Immunopathol. 1994;73:146-151.Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med. Feb 12 2009;360(7):711-720. McCarty, G.A., Valencia, D.W., and Fritzler, M.J., Antinuclear Antibodies-Contempory Techniques and Clinical Application to Connective Tissue Disease. New York: Oxford University Press, Inc. 1984. Murray DL, Homburger HA, Horvat RT, Snyder MR, College of American Pathologists; S-C 2009: Antinuclear Antibody Screening Methods; CAP Surveys S-C Diagnostic Immunology;2009 Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A. J.Clin.Pathol. 1999;52:684-687.Singer, M. and Berg, P., Genes & Genomes-A Changing Perspective. Mill Valley, CA: University Science Books. 1991.Sullivan KE. The complex Genetic Basis of Systemic Lupus Erythematosus, Reprint from 1999 and 2000; Lupus Foundation, Available at http://www.lupus.org/education/articles/geneticbasis.html Accessed June 16, 2010.Wallace DJ. New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability? Nat Clin Pract Rheumatol. Aug 2006;2(8):410-411.Willcocks LC, Carr EJ, Niederer HA, et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc Natl Acad Sci U S A. Apr 27 2010;107(17):7881-7885.	View Page

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