Transcript of Anti-nuclear antibody detection by IFA. Purpose Indirect Immunofluorecence Kits - Anti-nuclear...
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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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