بسم الله الرحمن الرحيم

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

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

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

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.

Many autoantibodies are associated with autoimmune diseases 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

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

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)

IMMUNOFLUORESCENCE DETERMINATION OF AUTOANTIBODIES: Detection of autoantibodies reacting with 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)

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

1-Antinuclear antibodies (ANA)

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 levels may correlate with intensity of inflammation Healthy persons may have low levels of ANA

mitotic apparatus centromere ANA ENA: extractable nuclear antigens

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

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

IIF-ANA test Materials Specimen Collection 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

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.

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.

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.

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.

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.

Y Y DETECTION OF AUTOANTIBODIES BY INDIRECT IMMUNOFLUORESCENCE NO NO 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 Y NO SERUM (AUTO Ab) WASHING Y AUTO Ag NO WASHING CELLS or Tissue GLASS slide

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.

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

Patterns Peripheral or rim Homogenous Speckled Centromeric Cytoplasmic Nucleoli

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

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

METHODS immunoblot ELISA recombinant or purified antigens

CLINICAL APPROACH TO ANA TESTING ANA by IFA Negative Positive Repeat anti-ds DNA anti-Sm SS-A(Ro) SS-B(La) U1 RNP (titer) ELISA Negative (stop) Positive Clinical Hx/PE (+) supportive AutoAb results

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)

2- Rheumatoid Factors (RF) & Related antibodies

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)

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

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- C1 -S-S- -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

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

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.

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

Procedure If +ve Semi-quantitative test (Serial Dilutions) 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)

Incidence of RF in Rheumatic Diseases 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 7 6

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

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.

3- Anti-Neutrophil Cytoplasmic Antigen (ANCA) antibodies

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

ANCA ELISA proteinase-3 myeloperoxidase

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

4- Antiphospholipid antibodies (APLA)

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.

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

4- ORGAN-SPECIFIC AUTOANTIBODIES

ANTI- AGAINST GASTRIC PARIETAL CELLS (GPC) Abs antigen: antigen H+/K+/ATP-asa clinical association: pernicious anemia (80-90%), autoimmune endocrine diseases,

Anti-Liver-Kidney Microsomal (LKM-1) Anti Mitochondrial Abs antigen: cytochrome mono-oxygenase mitochondrial antigen clinical association: markers of autoimmune hepatitis

ANTI-RETICULIN Abs (R1) antigen: reticulin R1 clinical association: Coeliac disease

ANTI-ENDOMYSIUM Abs (EMA) antigen: tissue transglutaminase clinical association: Coeliac disease

ANTI-SMOOTH MUSCLE Abs(ASMA) antigen: cytoskeletal antigens clinical asociation: marker of autoimmune hepatitis I, inflammatory diseases

Common Auto-antibodies in some autoimmune diseases Specfic Autoantibody Disease Anti-ds DNA & Anti-Sm SLE Anti Histone drug-induced lupus Anti-Jo-1 polymyositis Anti-U1-RNP Mixed Connective Tissue Disease Anti-Smooth Muscle, Anti-LKM autoimmune hepatitis Anti-CCP Rheumatoid Arthritis Anti-Scl-70 Scleroderma ANCA Autoimmune vasculitis

THE PRESENCE OF AUTOANTIBODIES HAS TO BE INTERPRETED SUMMARY: THE PRESENCE OF AUTOANTIBODIES HAS TO BE INTERPRETED IN CLINICAL CONTEXT

Acute phase proteins Inflammatory markers Produced mostly by hepatocytes Acute phase response: is the increase of serum proteins in response to cytokine release (esp. Il-1& IL-6) from monocytes and macrophages in inflammatory states Examples: CRP: Precise function unknown; activates complement, but also anti-inflammatory Rapidly fluctuates in response to inflammation Others: serum amyloid A (SAA), ferritin

Study question Mention the methods used to determine Anti-nuclear antibodies

Assignment Write shortly on the methods used to determine Anti- nuclear antibodies (ANA).

Thanks