Transcript Anti-dsDNA

‫بسم هللا 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: [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
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
Higher in women, elderly
5%
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 anticytoplasmic Antibodies
Ethanol and methanol fixation may remove Ro/SSA antigens
from cells, so the cells are fixed with acetone
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
PROCEDURE:
1.
2.
3.
4.
5.
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.
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.
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.
WASHING
ANTISERUM AGAINST HUMAN Ig
WASHING
Y
Y
WASHING
SERUM (AUTO Ab)
NO
WASHING
AUTO Ag
CELLS or Tissue
GLASS slide
NO
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 Hep2 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
Nucleoli
Speckled
Centromeric
Cytoplasmic
Anti-dsDNA
(native DNA)
kinetoplast formed
by dsDNA.
.
antigen:
Substrate
is protozoon
Crithidia luciliae,
with kinetoplast
formed by
dsDNA.
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
ELISA
recombinant or purified antigens
immunoblot
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:
1. Rheumatoid factors (RFs)
2. Anti-keratin antibody (AKA)
3. Anti-preinuclear factor
4. Anti-cyclic citrulinated peptide (Anti-CCP)
antibodies.
5. ANA (low titer)
1-Rheumatoid Factor (RF)
 RFs are auto-antibodies against Fc fragment of selfIgG
 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
C domains
C1
C2
C3
Agal IgG
Asn 297
His 435
binding
sites
for RF
GA – specific RF
IMMUNOPATHOGENESIS OF RHEUMATOID ARTHRITIS
deposition of immunocomplexes
to joint synovia
binding of RF to IgG
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
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
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
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
Wegener,s granulomatosis (c-ANCA)
Generalized
Active
Full remission
Localized
Active
Full remission
Polyarteritis group (p-ANCA)
Polyateritis nodule
Churg-Strauss syndrome
Polyangitis overlap syndrome
Idiopathic crescentic glomerlonephritis
% Positive
96
41
67
32
50
50
75
100
Inflammatory Bowl Diseases (Atypical)
70-82
Normal Indiveduals
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
Disease
Specfic Autoantibody
SLE
Anti-ds DNA & Anti-Sm
drug-induced lupus
Anti Histone
polymyositis
Anti-Jo-1
Mixed Connective Tissue Disease
Anti-U1-RNP
autoimmune hepatitis
Anti-Smooth Muscle, Anti-LKM
Rheumatoid Arthritis
Anti-CCP
Scleroderma
Anti-Scl-70
Autoimmune vasculitis
ANCA
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 Antinuclear antibodies (ANA).