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The most common method of ANA testing is indirect fluorescent assay (IFA) utilizing fluorescein isothiocyanate (FITC) as the marker on the secondary antibody.The fluorescent ANA test uses the indirect fluorescent antibody technique first described by Weller and Coons in 1954. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed.After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody conjugated to fluorescein. When results are positive, a stable three-part complex forms, consisting of fluorescent antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized with the aid of a fluorescent microscope. In positive samples, the cell nuclei will show a bright apple-green fluorescence with a staining pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear fluorescence. The cytoplasm may demonstrate weak staining while the non-chromosome region of mitotic cells demonstrates brighter staining.The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)

Alternative to the slide based methods for ANA detection include ELISA based assays as well as multiplex addressable laser bead immunoassays (ALBIA) such as the Luminex® platform. In these assays, selected antigens and/or a cell extract is coated into the wells of an ELISA plate or onto beads. These assays offer the convenience of automation, however, they lack the broad range of antigens that are present in the cells that are used as the substrate in the slide-based assay. This limited range of antigens results in false negative results for some samples that contain clinically significant ANA's.(Ref8) Position papers from such organizations as the American College of Rheumatology and the College of American Pathologists have called into question the accuracy of these automated ANA assays and recommend against using them as ANA screens.(Ref9) They further suggest laboratories inform the clinician as to which method the lab is using so they can better understand the results.

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.

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.

This sample is demonstrating an anti-cytoskeletal pattern in the cytoplasm of the cells.The cytoskeleton of eukaryotic cells is comprised of microfilaments such as actin, intermediate filaments such as vimentin and microtubules such as tubulin. Autoantibodies to these proteins are seen in a variety of autoimmune diseases.Whenever an ANA is read the first step is to see if there is a clearly discernible pattern in the nucleus of the interphase cells. In this case the answer is no (a). However, there is considerable staining in the cytoplasm. This fibrous staining of the cytoskeleton is a discernible cytoplasmic pattern and should be reported (b).This sample is reported as ANA Negative, suspect cytoskeletal antibodies present. (The term "suspect" is used because the ANA substrate is not considered "confirmatory" for the identification of cytoskeletal antibodies. Additional follow-up testing is required).Follow-up testing would include testing for anti-smooth muscle antibodies. Anti-smooth muscle antibodies are seen in autoimmune liver disease.Reporting anti-Cytoskeletal Antibodies:Cytoplasmic pattern:• Fibrous strands in cytoplasmReport as:• Suspect cytoskeletal, suggest follow-up Clinical Significance:• Actin: Autoimmune hepatitis, PBC• Vimentin and others: SLE, RA, others

Substance Stain or DyeCommentsProteinsPonceau SCoomassie Brilliant BlueSilverSpecific for ProteinsSilver is a biohazardLipoproteinsSudan Black BOil Red O-EnzymesEnzyme substrate and achromagen or fluorescent dyeReaction catalyzed by enzyme and color or fluorescence detectedHemoglobinNot neededIs intensely coloredNucleic Acids (DNA/RNA)Ethidium Bromide (EtBr)SyBr GreenSilver EtBr is CarcinogenicSyBr Green is new - Introduced in 1995Silver is a biohazard

Detection techniques can vary in both direct and amplified methodologies and can include labeling either the probe or the target molecule of interest:Chemiluminescence: Release of light energy at the end of a chemical reaction that is detected by a luminometer; uses a label such as acridinium ester. Electrophoresis: movement in a matrix such as a gel that is caused by an electrical field.Enzyme: Uses enzyme and substrate principles to label the appropriate target or probe; can be combined with fluorescence or dyes for detection.Fluorescence: Molecules that emit light at a longer wavelength when excited at a shorter wavelength. Detection techniques include fluorescent staining of nucleic acids as well as fluorescent labeled probes that are measured in a fluorometer or with fluorescent polarization.Radioactivity: Uses a labeling technique where the radioactive label is then measured in a scintillation counter. The earliest assays utilized radioactive decay.

HbSS blood may contain reticulocytes with an abnormal presence of CD36 on their membranes, allowing platelets to form a bridge between these young sickle cells and endothelial cells in post-capillary venules. This initial slow down of blood flow creates an environment in which cells containing HbSS can easily form sickled cells.The rigid cells, which are formed as a result of the sickling process, can collect in and plug small blood vessels. This can then cause tissue damage and organ infarction. In addition, the polymerized hemoglobin is thought to disrupt the RBC membrane, exposing phosphatidylserine that can trigger hemostasis. Subsequently, white blood cells (WBCs) may adhere to endothelium in response to recruitment in the inflammatory process. This leads to further occlusion as neutrophils capture additional RBCs in the post-capillary venules.Contributing further to vaso-occlusion is the decreased level of L-arginine in patients with sickle cell disease. L-arginine is a substrate needed to produce nitric oxide (NO). NO has vasodilatory properties as well as anti-inflammatory and anti-platelet properties. Thus a decrease in NO may lead to increased cellular adherence to endothelium.

A chemiluminescence substrate is added. A light-producing chemical reaction occurs between enzymes and substrates and a luminometer measures the light.

Reverse transcriptase PCR (RT-PCR) was developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). Essentially, the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR.PCR depends on the Taq Polymerase enzyme; RNA is not an efficient substrate for this enzyme. This is why the target of interest (if present) is first transcribed into complementary DNA (cDNA), which can then be amplified. RT-PCR ProcessAfter RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest, and nucleotides.If the target is present, primers anneal to the RNA strand.Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.The temperature is raised to 95o C, and the RNA/DNA strands are denatured.The temperatures are lowered, allowing primers to anneal to the newly formed cDNA.Polymerase enzyme synthesizes a new DNA strand, extending from the primer.Multiple cycles geometrically increase the number of copies of DNA.RT-PCR can be performed as one or two step procedures. In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction. In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40o C and 50o C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate reaction.

In order for hematein to attach itself to the nuclear components within a cell, a mordant must be present. A mordant is a chemical that serves as a link between the dye and the substrate. The result is an insoluble compound that helps adhere the dye to the cells.The most useful mordants for hematoxylin are salts of aluminum, iron, tungsten, and occasionally lead. These are classified respectively as : Alum hematoxylins Iron hematoxylins Tungsten hematoxylins Lead hematoxylins The type of mordant used influences the type of tissue components stained and the final color.Most mordants are combined into the staining solution, although a pre-treatment with the mordant is required with some hematoxylin stains.