الله بسمالرحيم الرحمن

Faculty of Allied Medical Sciences

Clinical Immunology & Serology Practice

(MLIS 201)

AUTOANTIBODIES IN THE DIAGNOSIS

OF AUTOIMMUNE DISEASES

Prof. Dr. Ezzat M HassanProf. of ImmunologyMed Res Inst, Alex UnivE-mail: elgreatlyem@hotmail.com

OBJECTIVES

1. To understand the concept of auto-immunity

2. To define auto-antibodies

3. To know the classes of autoimmune diseases

4. To know how to detect auto-antibodies

5. To know the antibodies in rheumatic diseases

6. To define the ANA and their patterns

7. To describe different methods for detection ANA and how to interpret the results

8. To know different auto-antibodies in rheumatoid arthritis, methods of detection and their clinical importance

9. To describe ANCA and anti-phosphlipid antibodies and their clinical significance

10. To define some-organ specific auto-antibodies

11. 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.

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

Results of loss of autotolerance

Results in inflammation

Autoimmune diseases are either:

Organ specific or

Organ non-nespecific (systemic)

Autoimmunity

LABORATORY DETERMINATION OF AUTOANTIBODIES: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:

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

ENA: extractable nuclear antigens

mitoticapparatus

centromere

ANA

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 ANADetection 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 TESTMaterialsSubstrate slide

Hep-2 (human epithelial cells)Positive Control

Specific autoantibody activityNegative Control

Pooled normal human seraUniversal FITC conjugate

Fluorescein conjugated to AHGMounting media

polyvinyl alcoholPhosphate buffered saline Evans Blue counter stain

Specimen CollectionSerum (recommended)

Avoid grossly hemolyzed or lipemic samples produce increased nonspecific background staining of the subtrate

PROCEDURE:1. Bring all reagents, materials and patient specimens to ambient

temperature. (Approximately 20 min)2. Construct a humidity chamber.3. Prepare a 1:40 dilution (5µl serum: 200 µl PBS) for each

specimen.

4. Remove slide from foil bag. Place in humidity chamber. Immediately add 25 l controls or diluted test serum to the appropriate wells.

5. Cover humidity chamber. Incubate slides @ ambient temperature for 30 minutes.

PROCEDURE:CONT.

6. Rinse each slide briefly with a stream of PBS.

7. 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.

8. 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.)

9. Place slides in humidity chamber and immediately dispense 25 l of FITC conjugate into each well.

10.Cover humidity chamber & Incubate for 30 minutes @ ambient temperature in the dark. Exposure to light will cause quenching of the fluorescent stain.

PROCEDURE:CONT.11.Rinse each slide briefly with a stream of PBS

12.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.

13.Remove slides, blot, and apply 4 1 drops of mounting media per slide, making sure to cover all wells.

14.Add coverslip.

15.View under fluorescent microscope in a dark room.

16.Evaluate each well for fluorescent staining

using +ve & -ve control wells as

a reference.

YY

GLASS slide

AUTO Ag

SERUM (AUTO Ab)

FLUOROCHROME - CONJUNG.

ANTISERUM AGAINST HUMAN Ig

NO

NOWASHING

WASHING

WASHING

WASHING

POSITIVE RESULT (AUTO Ab+)

NEGATIVE RESULT (AUTO Ab-)

UV LIGHTLIGHT EMISSION(FITC GREEN) UV LIGHT NO EMISSION

CELLS or Tissue

DETECTION OF AUTOANTIBODIES BY INDIRECT IMMUNOFLUORESCENCE

DETECTION OF AUTOANTIBODIES BY INDIRECT IMMUNOFLUORESCENCE

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 = dsDNAHomogenous = dsDNA,

histonesSpeckled = RNPNucleoli = Ku Antigen Centromeric = KinetochoreCytoplasmic = Jo-1 (RNA

synthetase)

PATTERNS

Peripheral or rim Homogenous Speckled

Nucleoli Centromeric Cytoplasmic

.antigen: native dsDNA

clinical association: Significantly (95%) associated with SLE Correlated with disease activity

Anti-dsDNA (native DNA)

Substrateis protozoonCrithidia luciliae, with kinetoplast formed by dsDNA.

kinetoplast formed by dsDNA.

2- By molecularly characterized antigens

• Targets (antigens) are produced by biotechnology

• Methods:

ELISA tests

immunobloting

Detection of ANA (cont.)Detection of ANA (cont.)

METHODS

immunoblotELISA

recombinant or purified antigens

TO SUMMARIZE…

Screen for ANAs using IIF on slides with HEp-2 cellsIf 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

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:

1. Rheumatoid factors (RFs)2. Anti-keratin antibody (AKA)3. Anti-preinuclear factor4. Anti-cyclic citrulinated peptide (Anti-CCP)

antibodies.5. ANA (low titer)

Rheumatoid Arthritis

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

1-Rheumatoid Factor (RF)

-S-S-

-S-S- -S-S--S

-S-

-S-S

-

-S-S

-

-S-S

-

-S-S-

-S-S-

-S-S-

-S-S--S-S-

-S-S--S-S-

-S-S--S-S-C1

C2

C3

V domains

C d

omai

ns

bindingsites

for RFHis 435

Asn 297

GA – specific RF

Agal IgG

MOLECULAR TARGETS FOR RHEUMATOID FACTORSMOLECULAR TARGETS FOR RHEUMATOID FACTORS

binding of RF to IgG

IgG

RF

deposition of immunocomplexes to joint synovia

IMMUNOPATHOGENESIS OF RHEUMATOID ARTHRITISIMMUNOPATHOGENESIS OF RHEUMATOID ARTHRITIS

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.

1-Rheumatoid Factor (RF) Cont.

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

RF Latex Agglutination Test

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 slideAdd 1 drop of well shaken latex reagentMix the two drops together with a clean stirrer and spread out to the

edge of the test areaRock the slide gently and observe for macro-agglutinationRead at 2 minutes under a direct light sourceA definite clumping is reported as reactive (R). No clumping is reported as non-reactive (N).

If +ve Semi-quantitative test (Serial Dilutions)

70-90

58

18

16

13

10

10

7

6

6

70-90

58

18

16

13

10

10

7

6

6

Rheumatoid Arthritis

Sjogrens,s syndrome

Systemic lupus erythematosus

Dermatomyositis

Mixed connective tissue disease

Cranial arteritis

Polymyalgia rtheumatica

Polyarthritis

Scleroderma

Juvenile rheumatoid arthritis

Rheumatoid Arthritis

Sjogrens,s syndrome

Systemic lupus erythematosus

Dermatomyositis

Mixed connective tissue disease

Cranial arteritis

Polymyalgia rtheumatica

Polyarthritis

Scleroderma

Juvenile rheumatoid arthritis

% Incidence% IncidenceDiseaseDisease

Incidence of RF in Rheumatic DiseasesIncidence of RF in Rheumatic Diseases

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

proteinase-3 myeloperoxidaseELISA

Diagnostic Specificity of (ANCA)Diagnostic Specificity of (ANCA)

9641

6732

505075100

9641

6732

505075100

Wegener,s granulomatosis (c-ANCA) Generalized Active Full remission Localized Active Full remissionPolyarteritis group (p-ANCA) Polyateritis nodule Churg-Strauss syndrome Polyangitis overlap syndromeIdiopathic crescentic glomerlonephritis

Inflammatory Bowl Diseases (Atypical)

Normal Indiveduals

Wegener,s granulomatosis (c-ANCA) Generalized Active Full remission Localized Active Full remissionPolyarteritis group (p-ANCA) Polyateritis nodule Churg-Strauss syndrome Polyangitis overlap syndromeIdiopathic crescentic glomerlonephritis

Inflammatory Bowl Diseases (Atypical)

Normal Indiveduals

Disease % Positive Disease % Positive

70-82

1-2

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.

28-7128-643827-3325-3120-40

28-7128-643827-3325-3120-40

Recurrent Venous Thrombosis

Recurrent Fetal Loss

Hemolytic Anemia

Thrombocytopenia

Arterial Occlusions

Pulmonary Hypertension

Recurrent Venous Thrombosis

Recurrent Fetal Loss

Hemolytic Anemia

Thrombocytopenia

Arterial Occlusions

Pulmonary Hypertension

% Incidence% IncidenceConditionCondition

Disease Association of Anti-Cardiolipin Antibodies

Disease Association of Anti-Cardiolipin Antibodies

4- ORGAN-

SPECIFIC

AUTOANTIBODIES

antigen: antigen H+/K+/ATP-asa

clinical association: pernicious anemia (80-90%), autoimmune endocrine diseases,

ANTI- AGAINST GASTRIC PARIETAL CELLS (GPC) Abs

antigen: cytochrome mono-oxygenase mitochondrial antigenclinical association: markers of autoimmune hepatitis

Anti-Liver-Kidney Microsomal (LKM-1)

Anti Mitochondrial Abs

antigen: reticulin R1

clinical association: Coeliac disease

ANTI-RETICULIN Abs (R1)

antigen: tissue transglutaminase

clinical association: Coeliac disease

ANTI-ENDOMYSIUM Abs (EMA)

antigen: cytoskeletal antigens

clinical asociation: marker of autoimmune hepatitis I, inflammatory diseases

ANTI-SMOOTH MUSCLE Abs(ASMA)

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

IN CLINICAL CONTEXT

SUMMARY:SUMMARY:

ACUTE PHASE PROTEINSInflammatory markersProduced mostly by hepatocytesAcute 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