ANA in autoimmunity by DR. ANAMIKA DEV

AUTO IMMUNE DISEASE , IMMUNOFLUORESCENCE , DIFFERENT PATTERNS IN IF

1. ANA IN AUTOIMMUNITY - Dr. Anamika . D

2. Autoantibodies Directed against C omponents of the cell surface , Cytoplasm components N uclear components

3. Antibodies to cell nucleus component ANA, anti- dsDNA , antibodies to extractable nuclear antigen ( ENA), (anti- Sm , anti-RNP) Antibodies to cytoplasmic antigens anti-SSA, ANCA Cell-specific autoantibodies lymphocytotoxic antibodies, anti- neurone antibodies, anti-erythrocyte antibodies, anti-platelet antibodies Antibodies to serum components antiphospholipid antibody,antiglobulin , rheumatoid factor

4. Anti-nuclear Antibodies A lso known as antinuclear factor or ANF 4 types: Anti- nuclear antibodies to DNA ANA to histones ANA to non-histone proteins bound to RNA ANA to nucleolar antigens

5. Many sub-types anti-Ro/SS-A antibodies Sjogrens syndrome anti-La/SS-B antibodies anti- dsDNA antibodies anti- Sm antibodies SLE anti- nRNP antibodies – Mixed connective tissue disease anti-Scl-70 antibodies - scleroderma Anti-Jo-1 antibodies – Polymyositis and dermatomyositis anti-histone antibodies – drug induced lupus anti-centromere antibodies – CREST syndrome anti-sp100 antibodies – primary biliary cirrhosis

6. ANA TESTING Three main methods: Indirect Immunofluorescence Assay (IFA) Enzyme-linked immunosorbent assay (ELISA) Multiplex Bead Immunoassays Microarrays

7. Sample preparation Collect blood specimens, usually from arm of pt , at any time. No special instructions given S eparate the serum. Specimens may be refrigerated at 2–8°C for up to seven days or frozen for up to six months. Avoid repetitive freezing and thawing.

8. Indirect immunofluorescence Immunofluorescence is commonly used In the past patients serum was placed on to slides with rodent (or other animal) cells and IF was performed to look for antibodies binding to cellular components What problems does this cause?

9. Human and rodent cells differ (slightly), and so some people with obvious rheumatic disease would be negative on this test. “ANA-negative lupus” Now there are human tumor cell lines that are used (HEp-2 are preferred)

10. How is the test done? Whole HEp - 2 cells containing these antigens are attached to microscope slide (cells fixed into separate dots on the slide ) Patient serum is diluted and dropped onto HEp-2 slides If antibody is resent , it binds to antigen on Hep -2 cells Incubate for 20 mins at room temerature . Wash to remove unreacted antibody. Add secondary antibody -anti-human globulin labeled with fluorescent tag or enzyme  bind to pts antibody  characteristic fluorescent pattern Read using an IF scope

11. Another source of false negatives includes how the tissues are fixed onto the slides Ethanol and methanol fixation may remove Ro/SSA antigens from cells, so the cells are fixed with acetone

12. Results D epend on Titer of antibody (highest dilution of serum at which auto- antibodies are still deectable ) IFpattern Titers less than 1:40 should be considered negative (20-30% of healthy people) Titers of 1:40 to 1:160  positive at low titer (further workup is not recommended in the absence of specific symptoms)

13. Results Titers equal to or greater than 1:160  positive

14. IF Patterns Peripheral or rim staining pattern = dsDNA Homogenous/ diffuse pattern = chromatin,dsDNA , histones Speckled = many antigens like Sm antigen, Ribonucleo -protein, SS-A& SS-B reactive antigens Nucleolar = RNA Centromeric = centromere

15. Ro La Smith RNP Jo-1 Scl-70 Ro Nucleolar dsDNA Rim Speckled Homogenous Nucleosomes Ro La Smith RNP Jo-1 Scl-70 Ro dsDNA Nucleolar proteins

16. Homogenous pattern Smooth, even staining of the nucleus with or without apparent    masking of the nucleoli Seen in Systemic lupus – anti – DNA, anti- histone

17. Rim/ Peripheral pattern Fluorescence is most intense at the periphery of the nucleus with a large ring starting from the internal nuclear membrane and the rest of the nucleus showing weaker yet smooth staining . Not seen on Hep-2 Seen in SLE – anti -DNA

18. Nucleolar pattern 23 or 46 (or some multiple of 46) bright speckles or ovoid granules spread over the nucleus of interphase cells Seen in Diffuse Scleroderma Scl-70

19. Speckled pattern Large speckles covering the whole nucleoplasm, interconnected by a fine fluorescent network. m/c observed Least specific Seen in sjogrens syndrome, SLE, polymyositis , dermtomyositis

20. Patterns

21. IFA- False Negatives: Rodent / animal cells used Methanol /ethanol fixation In Sjogren’s Syndrome,polymyositis , and dermatomyositis (ANA - ve in >50%) If single antibody, at very low level, is present : - SSA, subacute cutaneous lupus - dsDNA , ANA negative lupus

22. False Positives: 33 % of normals can be positive > 20% healthy relatives 75 % elderly population I n other diseases: viral infections, cirhosis , Chronic Pulmonary Fibrosis , Chronic Infection, Chronic Hepatitis ,Cancer

23. ELISA Also k/a ANA BLOT test or ANA ELISA TEST Amount of antibodies in units per given amount of blood Whole Hep-2 cells are lysed, centrifuged to concentrate the nuclei Other purified antigens are added, boost signal for all autoantigens (e.g., SSA, Scl-70, Jo-1) Coated onto high-affinity binding wells to retain all signals during processing

24. Diluted human serum is added to wells coated with purified nuclear antigens. ANA IgG specific antibody, if present, binds to the antigen. All unbound materials are washed away and the enzyme conjugate is added to bind to the antibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. The plate is incubated to allow the hydrolysis of the substrate by the enzyme . The intensity of the color generated is proportional to the amount of IgG specific antibody in the sample.

25. ELISA detects the same ANA antibodies as IFA, with improved sensitivity/specificity Results in one determination (no repeating to titrate) O bjective , automatable, requires no specialized training ELISA reports out Index Values

26. Multiplex Bead Immunoassay Individual antigens, cell components are coated onto multiple beads All activities are detected and identified in parallel High cost of test and automation

27. To summarize… You screen for ANAs using IF on slides with HEp-2 cells If it’s positive  look for the specific antigen using ELISA We don’t screen for ANAs using ELISA because it’s hard to get all the various antigens (40+) onto the well walls