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Anti-nuclear antibody detection by IFA

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Purpose Indirect Immunofluorecence Kits - Anti-nuclear antibody (ANA) HEp2 Cell ANA Kit - patterns

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Introduction What are antinuclear antibodies (ANA) ? - ANA are IgG antibodies against intracellular components of human cells. - ANA : (a) EIA (b) IFA (titer) (c) ELISA kit -

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Principle ANA (IFA) HEp-2 cells FITC conjugated antibody , FITC

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HEp2 cell Human pharyngeal cancer cell. Used as the nuclear substrate. Large nuclei, staining patterns can be observed in detail. Cells in various stages of the cell cycle coexist and antibodies to centromeres and PCNA (proliferating cell nuclear antigens) can also be detected.

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Materials HEp-2 Cell Substrate FITC conjugated goat anti-human immunoglobulin PBS Buffer wash & dilute serum Evans blue counterstain Positive Control Negative Control Mounting medium Cover slip

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Procedure Dilute serum 1:20 with PBS serum: positive control negative control serum x3(or 4) patient x2, 30~40l slide antigen well moisture chamber , 20~30min slide PBS staining basket, wash 5min ( stir bar) blotting paper well well

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slide moisture camber Slide well 1 drop secondary antibody Incubate for 20min at R.T. PBS wash, Wash , blotting paper slide 2~3 drops mounting medium, View slides under fluorescent microscope

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(-): No specific fluorescence is detected in nucleus. (): Although slight staining is detected in cell nucleus, staining pattern cant be identified. (+): Specific fluorescence is clearly detected throughout the entire nucleus, or in a certain area of nucleus. (-)

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(titer) kit: 1:20 positive Lab& : report as positive > 1:40 positive serum serial dilution

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ANA - Homogeneous pattern - Peripheral pattern - Speckled pattern - Centromere (Discrete speckled) - Nucleolar pattern - PCNA pattern - Others ex. mitochondrial cytoplasm

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Homogeneous pattern Chromosome region in a mitotic cell shows the same or brighter fluorescence than that in interphase period. Associated autoantibody: Anti-histone Disease Association: SLE Rheumatoid Arthritis

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Peripheral pattern In a mitotic cell, chromosome region is strongly stained Associated autoantibody: Anti-dsDNA Disease Association: SLE

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Speckled pattern The nucleoli are not usually stained The chromosome region in a mitotic cell is not stained Associated autoantibody: Anti-ENA Anti-SS-A Anti-SS-B Disease Association: SLE Sjogren's Syndrome

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Centromere(Discrete speckled) Homogeneously distributed and speckled fluorescence in the nucleus is observed. The number of speckles is 40~60 grains per nucleus. Mitotic figures have discrete speckles over the chromosome region. Associated autoantibody: Anti-CENP A, B, C, D Disease Association: Limited Scleroderma / CREST (60%)

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Centromere (Discrete speckled)

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Nucleolar pattern Nucleolus is stained as several large dots or clumps of granules inside nucleus. granules 6(per nucleus) Associated autoantibody: Anti-PM-Scl 4~6sRNA Disease Association: Extensive Scleroderma

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PCNA pattern Positive cells and negative cells coexist. Staining pattern varies depending on cell cycle. Associated autoantibody: Anti-cyclin Disease Association: SLE - often associated with glomerulonephritis

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Mitochondria Actin

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Mixed pattern Nucleolar plus speckled

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PatternCell TargetDisease Association Nuclear Homogeneous dsDNA SLE (95%) Discoid Lupus Autoimmune Hepatitis Rheumatoid Arthritis HistonesSLE NucleolarPM-Scl Extensive Scleroderma (30%) Polymyositis/Scleroderma overlap syndrome Primary Pulmonary Hypertension Nuclear MatrixhRNP Mixed Connective Tissue Disease SLE Speckled - fine SS-A (Ro) SS-B (La) Sjogren's Syndrome (95%) SLE (40%) Scleroderma (5%) Speckled - coursesnRNP SLE SLE overlap Syndromes CentromereCENT A-E Limited Scleroderma / CREST (60%) Raynauld's Phenomenon Proliferating Cell Nuclear Antigen (PCNA) CyclinSLE (1-3%) - often associated with glomerulonephritis Nuclear Dotsp80 coilinAutoimmune and viral liver disease

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PatternCell TargetDisease Association Cytoplasmic Fine SpeckledJo-1Polymyositis (20-40%) Ribosomal PatternRibosome PSLE (10-15%) MitochondrialM2 Primary Biliary Cirrhosis (90%) Scleroderma (40%) ER CytochromeP4 50 Drug Induced Hepatitis GolgiVarious SLE (rare) Sjogren's Syndrome Cytokeratin Rheumatoid Arthritis (30-80%) Mixed Connective Tissue Disease Autoimmune Hepatitis Crohn's Disease Squamous Cell Carcinoma of Lung

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Anti-neutrophil cytoplasmic antibodies (ANCAs) A group of mainly IgG antibodies against antigens in the cytoplasm of neutrophil granulocytes (the most common type of white blood cell). Major antigens associated with cANCA and pANCA are proteinase 3 (PR3) and myeloperoxidase (MPO) respectively.

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Cytoplasmic (cANCA) show a diffusely granular, cytoplasmic staining pattern Antigens: PR3 (90%), MPO (5%), & BPI (4%) This pattern results from binding of ANCA to antigen targets throughout the neutrophil cytoplasm, the most common protein target being proteinase 3 (PR3).

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Perinuclear (pANCA) show a perinuclear staining pattern Antigens: MPO (70%), lactoferrin (10%), elastase (8%), cathepsin G (5%) & PR3 (2%) This pattern occurs because during ethanol fixation some antigen targets artifactually localize around the nucleus. Antibody staining therefore results in fluorescence of the region around the nucleus.

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