بسم الله الرحمن الرحيم. faculty of allied medical sciences clinical immunology...
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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: [email protected]
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