بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

3. AUTOANTIBODIES IN THE DIAGNOSIS OF AUTOIMMUNEDISEASES Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com

4. Objectives • To understand the concept of auto-immunity • To define auto-antibodies • To know the classes of autoimmune diseases • To know how to detect auto-antibodies • To know the antibodies in rheumatic diseases • To define the ANA and their patterns • To describe different methods for detection ANA and how to interpret the results • To know different auto-antibodies in rheumatoid arthritis, methods of detection and their clinical importance • To describe ANCA and anti-phosphlipid antibodies and their clinical significance • To define some-organ specific auto-antibodies • To define the acute phase proteins

5. Introduction • The main function of the immune system is to discreminate between self and non-self antigens • This is maintained by a process called immune tolerance. • The break-down of tolerance will result in an immune response directed at one or more self (auto) antigens • This leads to an abnormal auto-immune response causing autoimmune diseases with the production of various auto immune effector components . • One of these effectors is the production of auto-antibodies.

6. Autoantibodies • Autoantibodies are immunoglobulins that bind to self antigens (autoantigens) • Autoantigens include self molecules in the nucleus, cytoplasm, and cell surface • Many autoantibodies are associated with autoimmune diseases • Autoantibodies can be detected in healthy persons (at low level) without clinical significance • Mechanisms how normal immunity change to autoimmunity are multiple

7. Autoimmunity • Results of loss of autotolerance • Results in inflammation • Autoimmune diseases are either: • Organ specific or • Organ non-nespecific (systemic)

9. LABORATORY DETERMINATION OF AUTOANTIBODIES: Based on immunochemical detection by: * agglutination * precipitation:* turbidimetry * immunobloting * ELISA * immunofluorescence determination of autoantibodies can be: * qualitative +ve/-ve * quantitative: * titer * arbitrary units * concentration (standard)

10. IMMUNOFLUORESCENCE DETERMINATION OF AUTOANTIBODIES: • Detection of autoantibodies reactingwith tissue antigens • by IIF test using : • *Tissue sections from different animal species • *Cell suspensions: • Hep-2 cell line (ANA) • Granulocytes (ANCA) • Crethidia Luciliae (Anti-dsDNA)

11. Autoantibodies and Rheumatologic Diseases: When and How to Use Laboratory tests

12. 1-Antinuclear antibodies (ANA)

13. Antinuclear antibodies (ANA) • Serologic hallmark of systemic autoimmune connective tissue diseases • React with cell nuclear apparatus; may bind DNA, RNA, nuclear proteins, nucleolar proteins & protein-nucleic acid complexes (RNP) • Their levelsmay correlate with intensity of inflammation • Healthy persons may have low levels of ANA

14. mitotic apparatus centromere ANA ENA: extractable nuclear antigens

15. ANA in rheumatologic diseases SLE (Systemic Lupus Erythematosus) Drug-induced SLE RA (Rhematoid Arthritis)(40%) Polymyositis/dermatomyositis (75%) MCTD (Mixed Connective Tissue Sisease) ANA in non-rheumatologic diseases (eg. Autoimmune thyroiditis, Autoimmune hepatitis, Hepatitis C) • Normal Population 5% • Higher in women, elderly

16. Detection of ANA 1- BY IMMUNOFLUORESCEINCE Using : * tissue Sections: Mouse liver & kidney * cell suspensions: * Hep-2 cell line (ANA) *Crethidia Luciliae (Anti-dsDNA) Immunofluorescence is commonly used to look for antibodies binding to cellular components : Anti-Nuclear, and also anti-cytoplasmic Antibodies Ethanol and methanol fixation may remove Ro/SSA antigens from cells, so the cells are fixed with acetone

17. IIF-ANA test Materials Substrate slide • Hep-2 (human epithelial cells) Positive Control • Specific autoantibody activity Negative Control • Pooled normal human sera Universal FITC conjugate • Fluorescein conjugated to AHG Mounting media • polyvinyl alcohol Phosphate buffered saline Evans Blue counter stain Specimen Collection Serum (recommended) Avoid grossly hemolyzed or lipemic samples produce increased nonspecific background staining of the subtrate

18. Procedure: • Bring all reagents, materials and patient specimens to ambient temperature. (Approximately 20 min) • Construct a humidity chamber. • Prepare a 1:40 dilution (5µl serum: 200 µl PBS) for each specimen. • Remove slide from foil bag. Place in humidity chamber. Immediately add 25 l controls or diluted test serum to the appropriate wells. • Cover humidity chamber. Incubate slides @ ambient temperature for 30 minutes.

19. Procedure:cont. • Rinse each slide briefly with a stream of PBS. • Wash slides for 10 minutes in a staining dish filled with PBS. Gently agitate staining dish several times or use a magnetic stirrer during the wash.

20. Procedure:cont. • Remove each slide from staining dish and blot excess PBS from around the wells using blotting strips. (N.B. Do not touch the strip to the substrate wells.) • Place slides in humidity chamber and immediately dispense 25 l of FITC conjugate into each well. • Cover humidity chamber & Incubate for 30 minutes @ ambient temperature in the dark. Exposure to light will cause quenching of the fluorescent stain.

21. Procedure:cont. • Rinse each slide briefly with a stream of PBS • Wash slides for 10 minutes in a staining dish filled with fresh PBS and 5-6 drops of Evan’s blue counterstain. During wash, turn on microscope. NOTE: This wash step should also be set up in a dark place. Exposure to light will cause quenching of the fluorescent stain.

22. Procedure:cont. • Remove slides, blot, and apply 4 1 drops of mounting media per slide, making sure to cover all wells. • Add coverslip. • View under fluorescent microscope in a dark room. • Evaluate each well for fluorescent staining using +ve & -ve control wells as a reference.

23. DETECTION OF AUTOANTIBODIES BY INDIRECT IMMUNOFLUORESCENCE POSITIVE RESULT (AUTO Ab+) NEGATIVE RESULT (AUTO Ab-) LIGHT EMISSION (FITC  GREEN) UV LIGHT UV LIGHT NO EMISSION FLUOROCHROME - CONJUNG. ANTISERUM AGAINST HUMAN Ig WASHING WASHING NO Y SERUM (AUTO Ab) WASHING Y AUTO Ag NO WASHING CELLS or Tissue GLASS slide

24. Interpretation of Results • The test for autoantibodies is NEGATIVE if no specific pattern of fluorescence is observed on the substrate. • The test for autoantibodies is POSITVE when the Hep-2 substrate shows a specific pattern of fluorescence.

25. Patterns • Peripheral or rim = dsDNA • Homogenous = dsDNA, histones • Speckled = RNP • Nucleoli = Ku Antigen • Centromeric = Kinetochore • Cytoplasmic = Jo-1 (RNA synthetase)

26. Patterns Peripheral or rim Homogenous Speckled Centromeric Cytoplasmic Nucleoli

27. Anti-dsDNA (native DNA) kinetoplast formed by dsDNA. Substrate is protozoon Crithidia luciliae, with kinetoplast formed by dsDNA. . antigen: native dsDNA clinical association: Significantly (95%) associated with SLE Correlated with disease activity

28. Detection of ANA (cont.) • 2- By molecularly characterized antigens • Targets (antigens) are produced by biotechnology • Methods: • ELISA tests • immunobloting

29. METHODS immunoblot ELISA recombinant or purified antigens

30. To summarize… • Screen for ANAs using IIF on slides with HEp-2 cells • If it’s positive look for the specific antigen that the antibody is reacting by using ELISA or immunoblot • Checking for anti-dsDNA antibodies only when the symptoms are suspicious of SLE AND the ANA is positive • Guidelines suggest that the only antibodies that need to be quantified are dsDNA (to predict a flare, and nephritis risk)

31. 2- Rheumatoid Factors (RF) & Related antibodies

32. Rheumatoid Arthritis • It is a chronic inflammatory disease affecting mainly the joints and may affect other connective tissues. • It is characterized by the presence of various circulating auto-antibodies including: • Rheumatoid factors (RFs) • Anti-keratin antibody (AKA) • Anti-preinuclear factor • Anti-cyclic citrulinated peptide (Anti-CCP) antibodies. • ANA (low titer)

33. 1-Rheumatoid Factor (RF) • RFs are auto-antibodies against Fc fragment of self-IgG • Most RFs are of IgM but IgG & IgA RFs are also observed • It is present in 70-90% of RA patients • High titer RF is assocciated with extra-articular complications and systemic vasculitis • It is an important diagnostic and prognostic parameter • Negative RF: No rule out RA

34. MOLECULAR TARGETS FOR RHEUMATOID FACTORS V domains -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- C1 -S-S- binding sites for RF Agal IgG C2 Asn 297 C domains -S-S- -S-S- C3 His 435 -S-S- -S-S- GA – specific RF

36. IMMUNOPATHOGENESIS OF RHEUMATOID ARTHRITIS binding of RF to IgG deposition of immunocomplexes to joint synovia IgG RF

37. 1-Rheumatoid Factor (RF) Cont. • Although RF can exist as IgG, IgM, IgA & IgE isotypes, the IgM-class RF is the main class identified by clinically available diagnostic tests. • RF is detected by : • 1- Qualitative & Semi Quantitative Tests: • Rose-Waller hemagglutination (RBCs coated with human IgG) • Latex agglutination (Latex particles coated with human IgG) • 2- Quantitative Tests: • ELISA, Turbidimetry and Nephelometry • Measurement of IgM or IgG RF By ELISA does not give significantly more clinical information than traditional tests such as the Rose-Waaler or latex agglutination tests.

38. RF Latex Agglutination Test It is the most common screening serological test for RA Sample collection: • Allow 2 ml blood to clot at room temp (Formation of clot must be complete) • Separate serum and store at 2-8 °C if used within 2-3 days or at -20 oC if used later

39. Procedure • Bring all reagents to room temp. • Prepare 1:20 dilution of test serum (50 μl of serum to 1 ml of glycine buffer) • Place 50 μl of diluted serum on to the test slide • Add 1 drop of well shaken latex reagent • Mix the two drops together with a clean stirrer and spread out to the edge of the test area • Rock the slide gently and observe for macro-agglutination • Read at 2 minutes under a direct light source • A definite clumping is reported as reactive (R). • No clumping is reported as non-reactive (N). • If +ve Semi-quantitative test (Serial Dilutions)

40. Incidence of RF in Rheumatic Diseases Disease % Incidence Rheumatoid Arthritis Sjogrens,s syndrome Systemic lupus erythematosus Dermatomyositis Mixed connective tissue disease Cranial arteritis Polymyalgia rtheumatica Polyarthritis Scleroderma Juvenile rheumatoid arthritis 70-90 58 18 16 13 10 10 7 6 6

41. 2-AKA • Highly specific for RA, occur in 40% of RA patients • Present in 1/3 of RF negative patients with RA • Detected at early stages, even before joint symptoms • Associated with more active &/or sever forms of RA AKA, by IIF, on rat oesophagus

42. 3- Anti-perinuclear factor Not used now due to low sensitivity and in-consistency of the substrate • 4- Anti-CCP (By ELISA) • IgG antibody against Cyclic Citrullinated Peptide (CCP) • Highly specific (96%) and moderately sensitive (78%) for RA. • Not found in other diseases (contrast to RF) • Detected in 70% of early RA. • Detected in 30-46% of sero-negative RA. • Should be a one time test, does not need to be repeated or followed • It is predictive of erosive disease.

43. 3- Anti-Neutrophil Cytoplasmic Antigen (ANCA) antibodies

44. Anti-Neutrophil Cytoplasma Antigens (ANCA) ANTIBODIES • Diagnostic for various systemic autoimmune vasculitis. *2 types By IIF Cytoplasmic (C-ANCA), & Peri-nuclear (P-ANCA) * substrate are ethanole or formalin fixed human granulocytes

45. ANCA ELISA proteinase-3 myeloperoxidase

46. Diagnostic Specificity of (ANCA) Disease % Positive Wegener,sgranulomatosis (c-ANCA) Generalized Active Full remission Localized Active Full remission Polyarteritis group (p-ANCA) Polyateritis nodule Churg-Strauss syndrome Polyangitis overlap syndrome Idiopathic crescenticglomerlonephritis Inflammatory Bowl Diseases (Atypical) Normal Indiveduals 96 41 67 32 50 50 75 100 70-82 1-2

47. 4- Antiphospholipid antibodies (APLA)

48. Anti-Phospholipids Antibodies (APLA) • Heterogeneous antibodies, against phospholipids • Cardiolipin and its co-factor, β2-glycoprotiens are primary antigens of phospholipids. • Anti-cardiolipin antibodies (ACA) are raised in various diseases especially SLE complicated with thrombotic manifestations. • They are measured in serum by ELISA • SLE patients who make anti-phospholipid antibodies will make VDRL test look like it’s positive because the VDRL particles are coated with phospholipids.

49. Disease Association of Anti-Cardiolipin Antibodies Condition % Incidence Recurrent Venous Thrombosis Recurrent Fetal Loss Hemolytic Anemia Thrombocytopenia Arterial Occlusions Pulmonary Hypertension 28-71 28-64 38 27-33 25-31 20-40

50. 4- ORGAN-SPECIFIC AUTOANTIBODIES