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Autoimmune

Disease Testing
Group 9
Fatima Tu Zahra
Komal Binte Ajmal
Maham Nadeem
Muhammad Ahmed
Saifullah khan
Sumayya Irfan

Autoimmune
Diseases
Ahmed

Autoimmunity

Immune System of body


Self antigens
Loss of immune homeostasis
Abnormal response against its own tissues

Hallmark of Autoimmunity
Self reactive T-cells
Autoantibodies
Inflammation

Laboratory Diagnosis
Cant be done by a single test
Several tests are needed to be done
Complete blood count
Comprehensive metabolic panel
Immunological studies
Serologies
Flow cytometry
Cytokine analysis
HLA typing
Disease activity
Erythrocyte sedimentation rate (ESR)

Initial Laboratory Evaluation

Blood analysis

Normochromic anemia
Platelet count
WBC count
Leukopenia and Thrombocytopenia common in SLE
patients

Serology
Abnormalities in serum level of specific enzymes
Abnormalities in metabolic processes
E.g. Autoimmune Hepatitis:

Elevated Transaminase
Elevated Bilirubin
Elevated serum proteins
Non-specific markers : Same in drug toxicity

Coagulation Studies
Antiphospholipid Syndrome
Prolongation of Activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT)
Inhibition of Clotting factor
Sarcoidosis
Hypercalcemia
Autoimmune Inflammatory Myopathies
(Dermatomyositis, Polymyositis and Inclusion body myositis)

Elevated creatinine kinase


Elevated ALT
Elevated AST

Urinalysis
Glomerulonephritis and Interstitial nephritis
Proteinuria
Hamaturia
Active sediment of RBCs and WBCs

All These Tests


Non-specific
But initial alarm for autoimmune disease

Ferritin
Komal Binte Ajmal

Ferittin
Iron binding protein
It sequesters iron in a non toxic and
readily available form

Critical component of iron


homeostasis

Hyperferitinemia
Hyperferitinemia is associated with inflammation due to
infection , malignancies or due to auto immune diseases

Though elevated levels are less often observed in cases of


auto immune diseases

Auto immune diseases


Rheumatoid arthritis
High concentrations of Ferritin are found in synovial fluid and
synovial cells or RA patients

SLE
Ferritin levels were high in the urine of patients with nephritis ,
pleural fluid and in the CSF of patients suffering from meningitis

Cont
THYRODITIS
Hormonal pathways are involved in the regulation of Ferritin
Elevated Ferritin levels
POLYMYOSTITIS /DERMATOMYOSTITIS
Inflammatory auto immune myopathy characterized by muscle weakness
and visceral movement

High Ferritin level

C reactive protein
Pentamic protein found in blood plasma
Synthesized by liver
Also an inflammatory marker
Detect inflammation which can be ether
due

to

diseases

infection

or

auto

immune

Significance Of CRP
The level of CRP in the blood is normally low.
A high or increasing amount of CRP in the blood suggests the presence of
inflammationbut will not identify its location or the cause. In individuals
suspected of having a seriousbacterialinfection, a high CRP can be
confirmatory. In people withchronicinflammatory conditions, high levels
of CRP suggest a flare-up or that treatment has not been effective.

If the CRP level is initially elevated and drops, it means that the
inflammation or infection is subsiding and/or responding to treatment.

DISEASES
RA
SLE
Vasculitis
LIMITATIONS
Non specific It only detects the presence of inflammation
but do not diagnose the cause

ESR
Commonly known as erythrocyte sedimentation rate
Simple , inexpensive but non-specific test
This test is used to measure the inflammation in body which can
either be a result of

1)infection
2)cancer
3)auto immune disorders

How the test is performed

The test measures how fast the RBCs fall to the bottom of a
thin long tube

The speed at which the RBCs fall to the bottom


In case of Inflammation, abnormal proteins appear in blood
which cause RBCs to clump together and make them fall
quickly

SIGNIFICANCE OF TEST IN
AUTO-IMMUNE DISORDERS
Elevated levels are shown which indicate inflammation
DISEASES
rheumatoid arthritis , SLE are most common

Limitations
Limitations of this test
It is a non specific test , it just indicates the presence of
inflammation but do not tell the exact location , cause and
diagnosis of disease

Inflammatory Markers
Maham Nadeem

Ceruloplasmin
Glycoprotein
Produced in liver
Carries or transports more than 95% of copper in
blood plasma
Plays a role iniron metabolism

Mechanism
Behaves as an enzyme (ferroxidase)
Catalyzes oxidation of Fe2+(ferrous iron) into Fe3+(ferric
iron)
Assists in transport in the plasma in association with
transferrin
Essential for normal movement of iron from cells to plasma

Periodontitis
A set of inflammatorydiseasesaffecting the tissues that
surround and support theteeth
Risk identified and quantified by biomarkers
Creates area of local inflammation indicated by elevated
serum levels of various pro-inflammatory markers
specially ceruloplasmin.

Increased levels in:

Inflammatory states
Pregnancy
Lymphoma
Rheumatoid arthritis
Alzheimer's disease

Haptoglobin
Protein
Encoded by theHPgene.
Produced in response to tissue injury

Mechanism
Inintravascular hemolysis, free

haemoglobin(Hb) released fromerythrocytes


Free Hb bound by haptoglobin
Haptoglobin-hemoglobincomplex formed
Phagocytosed by macrophages
Levels decline

In extravasclar hemolysis:
Macrophages phagocytose erythrocytes
Hb not released into circulation
No Hb-haptoglobin complex formed
Serum Haptoglobin levels rise

Increased levels in:

Inflammation
Malignancy
Surgery
Trauma
Peptic ulcer disease
Ulcerative colitis

Albumin
Serum protein
Synthesized by the liver
Main function is to regulate thecolloidal osmostic
pressureof blood
Inflammation results in a lower synthesis rate
Decreased serum concentrations (negative acute
phase protein

Mechanism
Distributed between the vascular and interstitial spaces
During inflammation, IL6 increases
Results in albumin being pulled from the intravascular spaces and
circulating to the liver until theinflammatory diseaseresolves
Liver starts producing other proteins needed to fight the
inflammation.
Inflammation simultaneously decreases albumin synthesis and
increases albumin catabolism.

Decreased levels in:

Autoimmune diseases
Infections
Malignancies
Trauma
Surgery, burn injuries
Crohns disease

Auto-antibodies
Saif

Auto-antibody
Auto anti-bodies are the proteins that are directed against

bodys own cells, tissues and organ causing inflammation


and damage.
Unknown causes of productions
Genetic predisposition and environmental triggers
Exposure to virus and toxins
Hormonal causes ( women are more susceptible)

Types of auto-antibodies

Rheumatoid Factor (RF)


RF binds to Fc portion of polyclonal IgG.
Helpful in evaluation of patients with Rheumatoid

Arthritis.
Specificity=70%
sensitivity=70%
Progressive joint morbidity and rheumatoid nodules.
Sjogren's syndrome, SLE, Cryoglobulinemia, interstitial
fibrosis and silicosis.
Diagnosis with ELISA.

Anti-citrullinated peptide
antibody.
Inflammation in RA.
Activation of peptidylarginine deiminase which incorporates
citrulline into certain proteins.
Antibody against CP.
Specificity= 95%
sensitivity= 70%.

RF/ACP antibody test


Both of these tests are primarily used as diagnostic tool
for Rheumatoid arthritis.
Procedure is same as that of ELISA.
Commercially available kits

Anti Nuclear Anti-bodies


Diverse group that react against nuclear, nucleolar and perinuclear

antigens
Hallmarks the serologic diagnosis of SLE.
Also present in some non-rheumatic disorders like Hashimoto and
Graves disease.
ELISA method.
Subtypes: anti-Ro antibodies,anti-La antibodies,anti-Sm
antibodiesanti-nRNP antibodiesanti-Scl-70 antibodies, anti-histone
antibodies, antibodies to nuclear pore complexes, anti-centromere
antibodies,andanti-sp100 antibodies.

Anti Nuclear Anti-bodies


Detection methods include ELISA and Indirect
immunofluorescence.
Indirect immunofluorescence
Primary antibody attaches to antigen
Secondary antibody with fluorophore binds to primary antibody
Fluorophore emits light when excited.
Amplification can be done using multiple secondary antibodies.

Anti-dsDNA antibody.
Diagnosis of Lupus nephritis, viral disease like parvo B19 and BK
virus.
Previously measured using radioimmunoassay (particularly the Farr
assay)
Nowadays measured using ELISA.
Immunofluorescence assay
target antigenCrithidia luciliae, a flagellated protozoa containing a dsDNAcontaining small organelle called a kinetoplast

The antibodies to dsDNA are detected semi-quantitatively by demonstrating IgG


bound to the kinetoplast.

Extractable
Nuclear Antigens
Anti-ENA testing
Sumayya

What are these?


Soluble Cytoplasmic and Nuclear

Antigens
Main antigens used in the laboratory for
disease detection are:
Ro
La
Sm
RNP
Scl-70
Jo1

Production of autoantibodies to ENA


are indicators of
specific diseased
conditions
associated with
many systemic
Rheumatic diseases
(SRD), including
Systemic Lupus
Erythmatosus (SLE),
Rheumatoid
Arthritis ( RA),
Sjrgrens

How are they measured?


Indirect Immunoflourescence
Elevated titers and staining pattern
indicates important results

Attention: The test lacks specificity and elevated


titers in individuals who do not have the disease
might be detected leading to false positive results
and referral to the rheumatologists

What do the results indicate?


High enough titer will be indicative of SRD
Presence of proteins in the situation when immune system
is unable to distinguish self from foreign.
Patients with positive ANA and symptoms of SRD are
recommended Anti-ENA tests
The signs and symptoms include: Inflammation of organs,
Muscle pain, Join pain and Fatigue

Anti-ENA Test: What does it


Measure?
Detects Auto-antibodies which specifically attack proteins in the
nucleus
The following auto-antibodies are detected:
Anti-Ro (for Sjrgrens Syndrome)
Anti-La (for Sjrgrens Syndrome)
Anti-Sm (for SLE)
Anti-RNP (for MCTD)
Anti-Scl-70 (for Scelrosis)
Anti-Jo1 (for Polymyostitis)

Ouchterlony double
immunodiffusion
Agar gel immunodiffusion or passive double
immunodiffusion
Immunological technique
Involved in Identification, Detection and Quantification

The Technique

Indirect Solid Phase Enzyme


Immunometric Assay
Anti-ENA Screen Kit

Principal: Anti-ENA
testing is based on the
binding of the antibodies
from the patient to the
antigens coated on a
microplate

Results

Refference: ENA Panel, Accessed at:


http://labtestsonline.org/understanding/analytes/ena-panel/tab/test/

Signal Recognition
Particle
Anti-Signal Recognition Particle Auto-antibodies

What is it?

Abundant
Cytosolic
Universally conserved ribonucleoprotein
They target and bind to specific proteins in the EPR

Anti-SRP auto-antibodies
Presence is associated with severe acquired necrotizing
myopathies
Characterized with elevated serum Creatine Kinase levels,
severe muscle weakness and poor response to traditional
immunoresponsive therapy
Diagnosis: 3-4 months

Why is it highly specific for


myositis?
It is extremely rare to find these antibodies for other
conditions of autoimmunity or muscle disease
5-8%of the patients with inflammatory myopathy have
anti-SRP antibodies
Strong evidence of the presence of the disease
Also indicate certain clinical patterns

The Anti-SRP antibodies


Directed against the SRP complex
Six subunits in the family associated with the a small RNA molecule
The patients anti-SRP sera may recognize one the six subunits or
7S RNA
SRP54- The Main Traget
Anti-SRP antibodies interfere the invitr translocation of the
secretory proteins in to the endoplasmic reticulum
CK levels are also elevated so their elevation is an important
indicator of Anti-SRP antibodies

Detection Methods
Indirect Immunoflourescence test (Not Specific)
Needs to be confirmed by an immunodot assay or protein
immunoprecipitation (Not Quantitative)

Immunodot Assay

Enzyme Immuno Assay


The antigens are spread as discrete dots on to a solid membrane
Specimen is added to a reaction vessel
The strip is inserted
The patients antibodies are allowed to bind to the antigens on the solid
membrane
The non-specifically bound material is removed
Alkaline phosphatase anti-Human antibodies are allowed to bind to the patients
antibodies
The strip is transferred to enzyme substrate reagent
Reaction with bound Alkaline phosphatase
Observance of a distinct dot

RNA Immunoprecipitation Assay

Anti-Neutrophil Cytoplasmic
Antibody

A group of auto-antibodies
Mainly, IgG type
They are agianst cytoplams of neutrophils and monocytes
Detected in a number of auto-immune disorders
Particularly associated with vaculitis

Immunoflourescence and ANCA


Done on ethanol fixed neutrophils
Formalin fixed may also be used
Help differentiate ANCA patterns

Difference between P-ANCA and CANCA

ANCA Antigens
c-ANCA
Protinase 3
P-ANCA
Myeloperoxidase
In addition p-ANCA has its antibodies against Bacterial
permeability increasing Factor (BPI), Cathepsin G,
Elastase, Lactoferrin and lysozyme.
Others: HMG1, HMG2, alpha enolase, catalase and actin
e.tc.

ELISA is also used for antibody


detection

The Complement System


It is a part of the immune system that is involved in
complementing the antibodies and the phagocytic cells to
clear the pathogens.
Has several triggers, when it is stimulated it directs
proteases that cleave specific proteins and release
cytokines that start an amplifying cascade.
Over 30 proteins and fragments make up this system

Its role in Diseases


It is involved in a number of autoimmune disorders
including, Arthritis, autoimmune heart disease, multiple
sclerosis
Deficiency in the terminal pathways contribute to these
diseases

Immunoglobulins

The body may start mistaking self antigens for foreign


So immunoglobulin test help detect autoimmune diseases
IgA, IgG and IgM are often measured together
Can be used to detect Juvenile idiopathic arthritis, lupus
and celiac diseases

Cryoglobulin
Flow cytometry
cytokine studies
MHC studies

Fatima Tuz Zahra

Cryoglobulin

Abnormal blood protein


Gamma globulins with a molecular weight of
approximately 200,000.
Precipitates blood serum when it is chilled
Binds with complement proteins and other peptides
to form immune complexes
Cause tissue damage

Types of Cryoglobulin
Type I
Monoclonal immunoglobulins
IgM isotype
Multiple myeloma
Waldenstrom's macroglobulinemia
Lymphoproliferative disorders

Types of Cryoglobulin
Type II
Mixture of polyclonal igG and monoclonal igm
Type III
Combination of polyclonal igG and polyclonal igM.
Hepatitis C
Small vessel vasculitis

How the Test is Performed


Blood is typically drawn from a vein, usually from the
inside of the elbow or the back of the hand
Cryoglobulins are very hard to detect accurately
Temperature sensitive

Detection Methods
If the cryoglobulin test is positive :
Protein electrophoresis
Immunofixation electrophoresis (IFE)

MHC/HLA

MHC class I and II genes


Genetic determinants of many autoimmune diseases
MHC class I molecules include HLA-A, -B, and C.
MHC class II molecules include HLA-DR, HLA-DQ, and
HLA-DP.
HLA class I and II have been associated with rheumatic
disorders

HLA studies
HLA-B27 is present in approximately 90% - 95% of white
patients with ankylosing
HLA-DR1 and HLA-DR4 increase the risk of polyarticular
juvenile idiopathic arthritis (JIA) in many populations.
HLA-DR3 and HLA-DR2 are associated with lupus in
Caucasian populations

Detection of HLA
Elisa
Gel electrophoresis
Polymerase chair reaction (PCR)

Cytokine studies
Cell signaling molecules that aid cell to cell
communication in immune responses
Stimulate the movement of cells towards sites of
inflammation, infection and trauma.
Cytokines that influence inflammation include il-1, il-6 and
tnf-alpha.
Detection
o DNA microarray studies

Cytokines Human Autoimmune


Diseases

Rheumatoid Arthritis
Systemic Lupus Erythematosus
Multiple Sclerosis
Psoriasis
Systemic Sclerosis

THANKS