autoimmune markers thesis

A Study of Autoimmune markers by Indirect Immunofluoresence Assay : ANA , ASMA , ANCA and dsDNA .

2016

Moiz Indorewala [Type the company name]

3/23/2016

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A STUDY OF AUTOIMMUNE MARKERS BY INDIRECT

IMMUNOFLUORESENCE ASSAY

Dissertation Report Submitted To The

University Of Mumbai

For The Partial Fulfillment of The Degree of

Master of Science In Biotechnology

(By Papers)

By

Mr. INDOREWALA MOIZ HATIM TASNEEM

Department of Biotechnology

Ramnarain Ruia College Of Arts & Science,

Matunga , Mumbai.

Under The Guidance of

DR. BARNALI DAS

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DECLARATION

I hereby declare that the dissertation entitled ‘A STUDY OF AUTOIMMUNE MARKERS BY

INDIRECT IMMUNOFLUORESENCE ASSAY’ is being submitted as partial fulfillment of the

requirements for the Degree of Masters of Science in Biotechnology and is not substantially the

same as one which has already been submitted for a degree or any other academic qualification

in any other university, institute or examination body.

Place: MUMBAI (MOIZ .H. INDOREWALA)

Date:

4

S.P.MANDALI’S

RAMNARAIN RUIA COLLEGE

MATUNGA (CR), MUMBAI – 400019.

DEPARTMENT OF BIOTECHNOLOGY

CERTIFICATE

This is to certify that Mr. Indorewala Moiz Hatim Tasneem has satisfactorily completed the

Research Project entitled ‘A Study of AutoImmune Markers Using Indirect

Immunofluoresence Assay’ as prescribed by the University of Mumbai towards the partial

fulfillment of the degree of M. Sc (Biotechnology) by papers during the academic year 2011 –

2012.

The work entered in this dissertation is the bonafide work of the student as carried out at

Kokilaben Dhirubhai Ambani Hospital and Research Institute.

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ACKNOWLEDGEMENT

Immeasurable appreciation and deepest gratitude for the help and support are extended to the

following people who in one way or the other have contributed in making this study possible.

Firstly, I would like to express my sincere gratitude to my research advisor, Dr. Barnali Das for

the continuous support, guidance, advises, provisions and immense knowledge that has helped in

the completion and success of this study.

I owe my deepest gratitude to all the staff members of the Immunology Department of Kokilaben

Dhirubhai Ambani hospital and Research Institute. Special thanks to Ms. Poonam Mandavkar,

along with Ms. Vishakha and Mr. Sachin who provided me an opportunity to join their team as

intern, and who gave access to the laboratory and research facilities.

I convey special thanks to the Principal, Dr. Suhas Pednekar and Miss. Supriya Kale, Head

of Department of Biotechnology, Ramnarain Ruia College of Arts & Science.

I thank my fellow trainees and friends for the stimulating discussions and the help and moral

support provided by them which made this activity effortless and enjoyable.

Last but not the least I would like to thank my parents for their immeasurable love and support

throughout the completion of my thesis and my life in general.

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ABBREVIATIONS

AID: AutoImmune diseases

ANA: Anti Nuclear Antibodies

ANCA: Anti Neutrophilic Cytoplasmic Antibodies

cANCA: Cytoplasmic Anti Neutrophilic Cytoplasmic Antibodies

pANCA: Perinuclear Anti Neutrophilic Citrullinated Antibodies

dsDNA: Double Stranded DNA

ASMA: Anti Smooth Muscle Antibody

AMA: Anti Mitochondrial Antibody

HLA: Human leukocyte Antigen

ESR: Erythrocyte Sedimentation Rate

RF: Rheumatoid Factor

Anti-CCP: Anti-cyclic Citrullinated Peptide antibody

Anti-ENA: Anti-Extractable Nuclear Antigen

Anti-SRP: Anti-Signal Recognition Particle

LAC: Lupus Anticoagulant

aCL: Anti-cardiolipin

aPL: Antiphospholipid auto antibodies

SLE: Systemic Lupus Erythematosus

ELISA: Enzyme Linked ImmunoSorbent Assay

HEp: Human Epithelioma

MCTD: Mixed Connective Tissue Disorder

WG: Wegener's Granulomatosis

MPA: Microscopic Polyangiitis

IBD: Inflammatory Bowel Disease

JIA: Juvenile Idiopathic Arthritis

PR3: Proteinase 3

MPO: Myeloperoxidase

CLIFT: Crithidia luciliae indirect immunofluorescence test

IF-ANA: Indirect Immunofluorescence Antinuclear Antibody Test

CTD: Connective Tissue Disorder

NuMA: Nuclear Mitotic Apparatus

CENP-F: Centromere Protein

ADCC: Autoimmune Diseases Coordinating Committee

NIH: National Institutes of Health

NIAID: National Institute of Allergy and Infectious Diseases

CDC: Centers for Disease Control and Prevention

FDA: Food and Drug Administration

IgA: Immunoglobulin A

IgG: Immunoglobulin G

IgM: Immunoglobulin M

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PBS: Phosphate Buffered Saline

nDNA : Native DNA

AIH: Autoimmune Hepatitis

PBC: Primary Billiary Cirrhosis

BEC: Billiary Epithelial Cells

LIST OF FIGURES

Figure

No.

Title Page No.

1 Diagrammatic representation of Crithidia luciliae homoflagellate

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2 Diagrammatic representation of common nuclear patterns observed under

fluorescence microscopy

14

3 Displays FY 2003 NIH funding by scientific category. 13

4 BIOCHIP coated with HEp-20-10 (Human epithelial) cells and primate

liver.(10 well)

14

5 BIOCHIP coated with granulocytes(EOH), HEp-2 cells+ granulocytes

(HCHO).(5well)

19

6 Kallestad® Mouse Stomach/Kidney, 8-well 19

7 Ds DNA slide 20

8 EUROIMMUN EUROStar 3Plus Immunofluorescence microscope with

LUCAM camera model# LU375C-10.

22

9 Fluorescence pattern ANA positive 23





14 Fluorescence pattern Anti-dsDNA positive 32


8


17 Fluorescence pattern ANCA positive 39






23 Fluorescence pattern ASMA positive 54





28 Fluorescence pattern AMA positive 75

29 Fluorescence pattern APCA positive

30 A butterfly rash typically seen in SLE

31 Juvenile Rheumatoid Arthritis

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ABSTRACT

INTRODUCTION: Autoimmune diseases are a family of more than 80 chronic, and often disabling, illnesses that

develop when underlying defects in the immune system lead the body to attack its own organs,

tissues, and cells. While many of these diseases are rare, collectively they affect 14.7 to 23.5

million people in this country, and – for reasons unknown – their prevalence is rising. Since

cures are not yet available for most autoimmune diseases, patients face a lifetime of illness and

treatment. They often endure debilitating symptoms, loss of organ function, reduced productivity

at work, and high medical expenses. This paper focuses on the detection of Autoimmune markers

in patients with different diseases and finding the clinical significance of the particular disease

and the autoimmune markers found in them during clinical diagnosis. The 4 markers presented in

this study are ANA, ANCA, dsDNA and ASMA. The method used here is indirect

immunfluoresence assay which is considered a gold standard for detection of autoimmune

markers

MATERIALS AND METHODS:

The method used here is indirect immunfluoresence assay which is considered a gold standard

for detection of autoimmune markers. The test detects the presence of ANA in the blood of the

patient which adhere to reagent test cells (substrate), forming distinct fluorescence patterns that

are associated with certain autoimmune diseases. EUROIMMUN IIFT kit was used for ANA and

ANCA whereas BIORAD Kallestad kit was used for Anti-dsDNA and ASMA. The slide

preparations were observed using fluorescence microscopy.

OBSERVATIONS AND RESULTS:

The 50 individuals included in the study were segregated according to the autoimmune markers

they were detected for. Among them 31 were found to be ANA +, 8 were found to be ANCA +,

and 12 were found to be positive for autoimmune liver disease (ASMA, AMA, APCA). The

primary dilution , intensity, pattern in case of ANA and the end point titer of the samples were

evaluated. The clinical history of the patients was obtained from the medical records department

of KDAH.

CONCLUSION: The clinical significance of autoimmune markers in various disease was determined. From this

we can say that autoimmune markers play an important role in defining and predicting a

particular disease by carrying out the sensitivity and specificity of prediction. The main topic of

discussion for this study is the mechanism of production or the reason for the presence of auto

antibodies in various diseases. These may not necessarily be autoimmune diseases, but the

presence of autoimmune markers in the serum makes for a good research topic.

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INTRODUCTION

utoimmune disorders occur when the body’s immune system turns against the body

itself, attacking as if it were a foreign pathogen. They comprise more than 50 distinct

diseases and syndromes, and affect about 5% of the population in, with two thirds of the

patients being female. Examples of autoimmune disorders include rheumatoid arthritis,

multiple sclerosis, juvenile diabetes, cardiomyopathy, antiphospholipid syndrome,

Guillain-Barré syndrome, Crohn’s disease, Graves’ disease, Sjogren’s syndrome, alopecia,

myasthenia gravis, lupus erythematosus, and psoriasis. (11) Examining patients for potential

autoimmune diseases is fraught with difficulty because not one laboratory test establishes such a

diagnosis. Typically, multiple laboratory tests are needed and include basic studies like a

complete blood count, comprehensive metabolic panel, acute phase reactants, immunologic

studies, serologies, flow cytometry, cytokine analysis, and HLA typing. Although some tests

may be non-specific, such as the erythrocyte sedimentation rate (ESR), they are useful to assess

disease activity. These tests can be useful in the diagnosis and management of patients with

autoimmune diseases and help in providing a prognosis, or indicate the severity of organ

involvement or damage.(4)

Historical perspective

Auto-antibodies against diseased organs in Sjogren’s sydrome were first described in 1965. The

mid-1960s also saw initial connections being made between viral infections and autoimmune

diseases, specifically hemolytic anemia, based on clinical observations By the early 1980s, there

were significant concerns about the links between bone marrow transplantation and autoimmune

disease. Ten years later, there was an interesting application of autoimmune disease principles in

a novel direction, namely that of birth control by immunization against sperm. (11)

The following research paper focuses on the clinical significance of 4 autoimmune

markers in various diseases. The 4 markers included in this study are –

1. Anti Nuclear Antibodies- ANA

2. Anti –Neutrophilic Cytoplasmic Antibodies- ANCA

3. Anti Smooth Muscle Antibodies- ASMA

4. Anti double stranded DNA- Anti-dsDNA

Some of the other autoimmune markers include Rheumatoid factor (RF) , Anti-cyclic

citrullinated peptide antibody (CCP), Anti-double stranded DNA (anti-dsDNA)

Anti-extractable nuclear antigen (anti-ENA), Anti-signal recognition particle (anti-SRP), anti-

JO-1, anti-Mi2, anti-PM/Scl, Lupus Anticoagulant (LAC)/anti-cardiolipin

(aCL)/antiphospholipid auto antibodies (aPL). (4)

A

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1. ANA

Auto antibodies to nuclear antigens are a diverse group of antibodies that react against nuclear,

nucleolar, or perinuclear antigens. These antigens represent cellular components such as nucleic

acid, histone, chromatin, nuclear and ribonuclear proteins. Classically, the ANA hallmarks the

serologic diagnosis of SLE, but finding an ANA is common to most other autoimmune diseases.

Methods used for detection utilize immunofluorescence testing of the patient's serum, at various

dilutions, using a cell substrate. Typically, screening patient's serum for the detection of an ANA

with ELISA provides high sensitivity but lacks specificity. Results are reported as either the

dilution of serum that tests positive or the degree of positivity measured by the testing procedure.

Historically, HEp20/10 cells (a human laryngeal epithelioma cancer cell line) have been used as

the cell substrate because the result offers the advantage of detecting a nuclear fluorescent

pattern. The fluorescent patterns (homogenous, diffuse, speckled, peripheral and rim) suggest

clinical

associations with certain autoimmune diseases. However, because of the time and expense for

testing with HEp2 cells, the assay procedures are largely done by ELISA methods.

Immunofluorescence is particularly useful as an initial screening test for those individuals

suspected of having an autoimmune disease – SLE, Sjögren's syndrome, RA, mixed connective

tissue disease (MCTD), scleroderma, polymyositis/dermatomyositis (PM/DM). However, one

must use caution when interpreting ANA as this autoantibody is found in nonrheumatic

diseases such as Hashimoto's thyroiditis, Graves' disease, autoimmune hepatitis, primary

autoimmune cholangitis, primary pulmonary hypertension, and in various infections.(4)

2. ANCA

Antineutrophil cytoplasmic antibodies (ANCA) react with cytoplasmic granules of neutrophils.

Initial ANCA testing screens sera for the presence of ANCA and two general immunofluorescent

staining patterns are observed – cytoplasmic (cANCA) and perinuclear (pANCA). The

immunofluorescence pattern is helpful to distinguish various ANCA associated vasculitis

syndromes. cANCA is most often seen in Wegener's Granulomatosis (WG), microscopic

polyangiitis (MPA) and Churg-Strauss syndrome. pANCA patterns were initially described in

microscopic polyangiitis (MPA), but pANCA has now been observed in a variety of diseases

including other types of vasculitis, inflammatory bowel disease (IBD), SLE, RA, juvenile

idiopathic arthritis (JIA). Antigenic determinants of the ANCA that are important to detect in

vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). Vasculitic states that test positive

are named PR3-ANCA positive or MPO-ANCA positive. The presence of PR3 or MPO can help

the clinician to determine the type of vasculitis and activity of disease.

Antibodies to PR3 or MPO are predictive of the various vasculitis syndromes. cANCA plus

PR3 have increased positive predictive value (PPV) for ANCA-associated vasculitis,

particularly WG. pANCA plus MPO has an increased PPV for MPA and less often for Churg-

Strauss. ANCA plus MPO more than PR3 often leads toward a diagnosis of Churg-Straus.

With increased disease activity, there is a greater likelihood that ANCA will be positive. ANCA

titers may normalize with treatment although persistent ANCA positivity or rising ANCA does

not reliably predict disease exacerbation or flare. Therefore, one should not use ANCA titers

to determine treatment efficacy. Furthermore, one should also be wary of ANCA positivity as

this can be seen in other disease states that include infection, drug use (eg. thyroid medication,

particularly PTU), and other autoimmune disease.(4)

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3. ASMA

Anti smooth muscle antibodies are proteins produced by the body’s immune system to work

against its own cytoskeletal proteins. The production of SMA is strongly associated with chronic

autoimmune hepatitis but may also be seen in other forms of liver disease and with other

autoimmune disorders such as biliary cirrhosis. Autoimmune hepatitis presents as an acute or

chronic inflammation of the liver that is not caused by another cause. It can lead to cirrhosis and,

in some cases, to liver failure.

ASMA and ANA tests are ordered to help diagnose and /or rule out other cause of liver injury.

These causes can include infections, drugs, alcohol abuse, toxins, generic conditions, metabolic

conditions and primary biliary cirrhosis

4. Anti dsDNA

A variety of methods has been developed over the years to measure antibodies to double

stranded (ds)DNA, a key diagnostic marker of systemic lupus erythematosus (SLE). These

methods include the Farr assay,ELISA other solid phase immunoassays, and the Crithidia

luciliae indirect immunofluorescence test (CLIFT). As current solid phase immunoassays have

variable performance due to lack of standardization, CLIFT is often regarded as a reference

method, because of its high clinical specificity. CLIFT uses the hemoflagellate, C. luciliae, as the

substrate. This protozoon, a single-cell organism, possesses a large modified mitochondrion,

called kinetoplast, containing a network of circular dsDNA. This network of dsDNA appears to

be free of histones or other mammalian nuclear antigens ( Therefore, reactivity against the

kinetoplast is specific for anti-dsDNA antibodies.

Fig 1. Diagrammatic representation of Crithidia luciliae homoflagellate

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The anti-double stranded DNA antibodies (anti-dsDNA) are considered a specific marker for

SLE.Due to the high frequency (ranging from 70% to 98%), sensitivity, and specificity (57.3%

and 97.4%, resp.), the presence of these auto antibodies could be virtually diagnostic for SLE.

Moreover, their identification in other pathological conditions and in healthy subjects is very rare

(less than 0.5%). Furthermore, the identification of anti-dsDNA in SLE patients several years

before disease onset suggests their involvement towards a clinically overt disease.

Indirect Immunofluorescence Antinuclear Antibody test (IF-ANA)

Although a battery of laboratory tests are available for ANA detection indirect

immunofluorescence antinuclear antibody test (IF-ANA) and enzyme immunoassay

(EIA)/enzyme linked immunosorbent assay (ELISA) are commonly used in day to day practice.

It is inexpensive and easy to perform, with high sensitivity and specificity [24]. The test detects

the presence of ANA in the blood of the patient which adhere to reagent test cells (substrate),

forming distinct fluorescence patterns that are associated with certain autoimmune diseases.

Initially different substrates like tissue sections, desquamated cells, chicken erythrocytes and

HeLa cells were tried but later on tissue sections using rat liver or a composite multiblock

substrate (mouse stomach, rat liver and kidney) became the standard substrate. In 1975 HEp-2

cells were introduced which have further increased the sensitivity of the test. These are the

cultured cells of laryngeal squamous cell carcinoma and are available commercially in the form

of prefixed on glass slides. Majority of the laboratories around the world are now using HEp-2

cell substrates. The correct interpretation of the IF-ANA results is important and must always be

correlated with the patient's symptoms and signs. While reporting IF-ANA three parameters are

evaluated; these include the pattern of fluorescence, substrate used and the titer of a positive test.

A negative IF-ANA result essentially excludes possibility of active CTD. Fluorescence patterns

and intensity squamous cell carcinoma and are available commercially in the form of prefixed on

glass slides The correct interpretation of the IF-ANA results is important and must always be

correlated with the patient's symptoms and signs. (1)

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Fluorescence patterns and intensity

Different staining patterns are reported which give clues as to the significance of the ANA and

type of CTD

1. Nuclear patterns: homogeneous, speckled (fine andcoarse), peripheral/rim, nucleolar,

centromeric, PCNA (proliferating cell nuclear antigen), nuclear dots, nuclear membrane, diffuse

grainy.

2. Cytoplasmic patterns: speckled, mitochondrial-like, ribosomal-like, Golgi apparatus,

lysosomal-like, cytoskeletal filaments (actin, vimentin, cytokeratin)

3. Mitotic patterns: mitotic spindle, centrosomes, NuMA (nuclear mitotic apparatus), midbody,

CENP-F (centromere protein)

Among these homogenous, speckled, peripheral and nucleolar patterns are more commonly

observed and of clinical importance. With any of these fluorescence patterns intensity of staining

with a qualitative scale of values from + to ++++ should also be reported as fluorescence

intensity is generally proportional to antibody concentration and predicts the severity of the

CTD.(1)

Fig2. Diagrammatic representation of common nuclear patterns observed under

fluorescence microscopy

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REVIEW OF LITERATURE One hundred years ago, renowned clinician William Osler observed that while some patients

died when their bodies were unable to fight off an infection, other patients appeared to die from

an excessive reaction by the body. Little was known about the human immune system at that

time, and it was not until 50 years later that scientists demonstrated convincingly that disease

could result not only from infection but from the body’s misguided attack on itself, even in the

absence of infection or other apparent cause. Today, we refer to this misguided attack as

autoimmune disease and understand that a delicate balance determines the difference between a

beneficial and injurious immune response.

More than 80 human diseases are due at least in part to an inappropriate immune system

response that results in damage to an individual’s organs, tissues, or cells. Autoimmune diseases

can affect any part of the body, and have myriad clinical manifestations that can be difficult to

diagnose. At the same time, autoimmune diseases share many features related to their onset and

progression. In addition, overlapping genetic traits enhance susceptibility to many of the

diseases, so that a patient may suffer from more than one autoimmune disorder, or multiple

autoimmune diseases may occur in the same family. While treatments are available for many

autoimmune diseases, cures have yet to be discovered. For these and other reasons, the

autoimmune diseases are best recognized as a family of related disorders that must be studied

collectively as well as individually.

Responding to the need for a concerted national effort to reduce the burden of

autoimmune diseases, the Autoimmune Diseases Coordinating Committee (ADCC) was

established by the National Institutes of Health (NIH) in 1998 and placed under the direction of

the National Institute of Allergy and Infectious Diseases (NIAID). The Children’s Health Act of

2000 further defined the Committee’s charge to expand, intensify, and coordinate research and

related NIH activities with respect to autoimmune diseases. The Committee is composed of the

directors, or their designees, of each of the Institutes and Centers involved in autoimmune

disease research; representatives of other Federal agencies, including the Centers for Disease

Control and Prevention (CDC) and the Food and Drug Administration (FDA), whose programs

include health functions or responsibilities relevant to these diseases; and representatives from a

number of private organizations concerned with autoimmune diseases.

Autoimmune diseases vary greatly in natural history and presentation. At the same

time, however, they seem to share many underlying immunologic mechanisms, and thus might

respond to similar treatment strategies. Over the past decade, more selective and less toxic

immunosuppressive and immunomodulatory agents have been developed to treat many of the

more than 80 autoimmune disorders so far identified. Now, scientists are using the growing

knowledge of the mechanisms of autoimmunity to develop promising approaches for inducing

immune tolerance, and are developing new and better strategies for early diagnosis, treatment,

and eventually prevention. They also are striving to strengthen the clinical research infrastructure

needed to test these new interventions.[26]

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Figure 3 displays FY 2003 NIH funding by scientific category. As in past years, the largest

expenditures were for fundamental studies of the genetic, environmental, and immunologic

factors underlying autoimmune diseases. Substantial investments were made for studies related

to diagnosis and disease progression, clinical research infrastructure, and conduct of clinical

trials. It is important to note that many projects focus on several scientific questions and are thus

difficult to place into a single category. For example, a training grant that focuses on training

scientists in epidemiology could be classified in two areas that are relevant to this figure.

Because projects were categorized according to their primary area of emphasis.[24]

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The recent assurance that indirect immunofluorescence (IIF) is the gold standard for anti-

nuclear antibody (ANA) testing from the Task Force of American College of Rheumatology

(ACR) has led clinical immunologists to revisit the urgent need for automated IIF platforms,

which could guarantee the robust and accurate interpretation of ANA patterns.

D. Roggenbuck et al. have provided an up-to-date overview of the data so far provided

using the AKLIDES system and critically discuss the pros and cons of digital automated indirect

IIF platforms for autoantibody detection in systemic rheumatological conditions.

J. Voigt et al. report original data on the evaluation of ANA on HEp-2 cells using the

EUROPattern Suite automated processing and interpretation system.This is the first time that

original data on the performance characteristics of this platformhave been published in the form

of a full-length paper. Concordant results between visual and automated evaluation by the

EUROPattern reached 99.4%.This supports the notion that a precise and reproducible

differentiation of positive and negative samples tested by HEp-2 cell lines is met by the

developed systems. Discrepancies betweenmanual and automated

pattern recognition is largely limited to serumsamples with mixed ANA patterns, but the

developers of those platforms assure the reader that they will soon overcome thecurrent

limitations.

J. Damoiseaux et al. evaluated the first automated anti-neutrophil cytoplasmic antibodies

pattern recognition system developed using the AKLIDES platform.Discrimination of C-ANCA

and P-ANCA is satisfactory but the sensitivity on ethanol-fixed neutrophils needs further

improvement. A considerable proportion of apparently healthy individuals have IIF-detected

ANA targeting the dense fine speckles 70 (DFS70) antigen. The clinical interpretation of positive

anti-DFS70 antibody associated pattern (DFS) tests has emerged as one of the most important

problems that routine laboratories are faced with, as it clearly influences the specificity and the

positive likelihood of the ANA tests.

In their review paper, M. Mahler and M. J. Fritzler discusstopic and also describe a

novel immunoabsorption method that can block anti-DFS70 antibody reactivity.

An original paper by M. Miyara et al. analyzed the clinical value of anti-DFS70

antibodies in patients subjected to routine ANA testing by IIF and concluded that systemic

autoimmune rheumatic disorders are less prevalent in patients with the DFS pattern compared to

patients with homogenous or other ANA IIF patterns.

R. Yoshimi et al. review the current data surrounding the clinical relevance and the

pathogenic significance of anti- Ro/SSA antibodies in systemic lupus erythematosus (SLE),

Sj¨ogren’s syndrome (SS), and other autoimmune disease.

An original paper by A. Wacker-Gußmann et al. provided data suggesting that foetal

magnetocardiography can complement foetal echocardiography as a noninvasive approach to

detect early electrophysiological signs of atrioventricular delay in foetuses exposed to maternal

anti-SSA/Ro and anti- SSB/La antibodies. Anti-C1q antibodies have been detected in women

with SLE who experienced failed pregnancy.

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An interesting study fromGreek investigators demonstrated that anti-C1q antibodies

cannot differentiate failed from normal pregnancies. The same investigators have found elevated

levels of IL-15 compared to those with missed abortions and healthy intrauterine pregnancies,

underlying the diagnostic potential of this marker.

Another original study has assessed the clinical relevance of circulating glucocorticoid-

induced TNFR-related protein ligand (GITRL) levels in patients with SLE.

Chinese investigators found that GITRL levels positively correlate with anti-dsDNA

titres and these levels were significantly higher in SLE patients with renal involvement and

vasculitis compared to patients lacking these clinical manifestations. Another original article

from a Chinese group has found that the titres of carbonic anhydrase III and IV auto antibodies

are unusually high not only in patients with SLE and rheumatoid arthritis, but also in patients

with other diseases including type 1 and type 2 diabetes.

P. C. Teixeira et al. review the literature and discuss the current knowledge on the role

of auto antibodies against apolipoprotein A-1 in cardiovascular diseases.

T. Shirai et al. provide an overview on the existing knowledge on the biological

significance of anti endothelial cell antibodies for vascular lesions in autoimmune rheumatic

diseases. They also discuss in great detail the principles and applications of identifying

autoantigens expressed on cell surfaces, known

as serological identification system for autoantigens using a retroviral vector and flow cytometry

(SARF).

A. Shimatsu and N. Hattori review the literature and discuss the diagnostic, clinical,

and pathophysiological features of macroprolactinemia caused by high molecular mass

complexes of prolactin with immunoglobulin G (IgG) and in particular anti-prolactin antibodies.

In their research article,

R. Fu et al. and co-investigators used a proteomic approach based on the membrane of

bone marrow cells to identify the antigenic targets of auto antibodies detected in a subgroup of

patients with immune-mediated pancytopenia.

A significant contribution comes from L. Mih´alyi et al. Those researchers provide a

meticulous overview of their 40- year long clinical experience in the diagnosis and management

of patients with autoimmune bullous dermatosis.

In their research paper, A. Patsatsi et al. found that titers of anti- BP180 auto antibodies

relate with disease activity in Greek patients with bullous pemphigoid, while a clinical study by

Dalm´ady and colleagues provided data suggesting that auto antibodies targeting mutated

citrullinated vimentin may

assist the diagnosis of psoriatic arthritis

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. U. Lindberg et al. found that IgA ANCA specific for bactericidal/permeabilityincreasing

protein (BPI-ANCA) identifies cystic fibrosis patients with adverse outcomesand discuss the

pathogenic potential of these auto antibodies.

. A. Antico et al. assessed the predictive value of combined testing of four serological

markers in the diagnosis of autoimmune gastritis. These markers include anti-parietalcell

antibodies (PCA), anti-intrinsic factor antibodies (IFA), anti-Helicobacter pylori (Hp) antibodies,

blood gastrin levels and are diagnostically useful in the classification of gastritis. Their predictive

value is comparable to that of histologically

assessed gastric biopsies

A British-German collaborative study found that the presence of Crohn’s disease-

specific pancreatic auto antibodies targeting the zymogen granule GP2 is largely limited to

patients with ileal involvement.

A.Kempinska-Podhorodecka et al. report on the influence of immunogenetics and

their close interaction with humoral markers of liver autoimmunity.These researchers found that

polymorphisms of genes involved in TNF-receptor signaling and in particular those of the TNF-

receptor-associated factor 1 (TRAF1) do not confer susceptibility to primary biliary cirrhosis

(PBC). However, the GG homozygotes have significantly higher titres of PBC-specific auto

antibodies directed against gp210 auto antibodies compared to AA homozygotes, suggesting that

this gene may immunoregulate the persistence of gp210-specific B-lymphocytes.

Another intriguing original paper by C. Radzimski et al. reports the development of a

recombinant cell based IIF assay which allows efficient determination of autoimmune hepatitis-

specific auto antibodies against soluble liver antigen.These auto antibodies are important for the

confirmation of the diagnosis in patients with suspected autoimmune hepatitis and cannot be

detected in routine laboratories by IIF.

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AIMS AND OBJECTIVES

Aim : To carry out the detection of autoimmune markers from the given patients samples using

indirect immunofluoresence assay and study its significance in the diagnosis of various diseases.

Objectives :

1. Collection of sample

2. Carry out IFA procedure

3. Obtaining patient’s clinical history

4. Finding out the interrelation of the autoimmune markers detected in the patient with the

clinical history obtained.

MATERIALS AND METHODS

This study was carried out at the Immunology department of Kokilaben Dhirubhai Ambani

hospital and Research Institute(KDAH), Andheri under the guidance of Dr. Barnali Das(MD,

DNB- Biochemistry, PGDHHM Consultant Biochemistry and Immunology) and Mrs. Poonam

Mandavkar(Technical executive). 50 individuals hospitalized during 16 November, 2015- 16

February, 2016 were selected from the clinical charts. Among them 18 were males and 32 were

females with an age range of 11-84 years. Prior to study recruitment, informed consent was

obtained from KDAH. As part of the study, each participant provided a blood sample and was

interviewed by trained personnel.

1. Sample preparation

5 ml of blood sample in clot activator tube

Centrifuged for 10 minutes at 4500 r.p.m to obtain serum.

Serum dilution and slide preparation.

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2.1 Slide preparation for ANA

Principle :

The EUROIMMUN IIFT Mosaic HEp-20-10/Liver(Monkey) kit is designed exclusively for the

invitro determination of human antibodies in serum or plasma.

Substrate combinations of Hep-20-10 cells and primate liver are incubated with diluted patient

sample. If the reaction is positive, specific antibodies of classes IgA, IgG and IgM attach to the

antigens. In a second step, the attached antibodies are stained with fluorescein-labelled anti

human antibodies and made visible with fluorescence microscope.

Requirements provided in the test kit :

i. Sample serum sample diluted 1:100 with PBS (10ul

serum+990 ul PBS)

ii. Slide BIOCHIP coated with HEp-20-10 (Human

epithelial) cells and primate liver.

iii. Conjugate fluorescein- labeled anti-human IgG (goat) ready

for use.

iv. Positive control auto antibodies against cell nuclei (ANA), control

serum with titre information, human, ready for

use.

v. Negative control autoantibody-negative, human, ready for use.

vi. Salt for PBS pH 7.2 -

vii. Tween 20 -

viii. Embedding medium Glycerol

Miscellaneous requirements:

i. Reagent trays

ii. Glass cover slips

iii. Test tubes and rack

iv. Pasteur and autopipettes

v. Coplin jars

vi. Distilled water

vii. Volumetric flask for PBS

viii. Wash bottles

ix. Foreceps

x. Timer

xi. Fluorescent microscope: EUROIMMUN EUROStar 3Plus Immunofluorescence

microscope with LUCAM camera model # LU375C-10.

22

2.2 Slide preparation for ANCA

Principle :

EUROIMMUN Granulocyte Mosaic and EUROPLUS Granulocyte Mosaic test kit is designed

for the qualitative or semiquantitative in vitro determination of human antibodies of

immunoglobin class IgG against granulocyte cytoplasm (ANCA).

Combinations of substrates are incubated with diluted patient samples. If the reaction is positive,

specific antibodies of classes IgA, IgG and IgM attach to the antigens. In a second step, the

attached antibodies are stained with fluorescein-labelled anti human antibodies and made visible

with fluorescence microscope.

Requirements provided with the test kit:


serum+90 ul PBS)

ii. Slide BIOCHIP coated with granulocytes(EOH), HEp-2

cells+ granulocytes (HCHO)

iii. Conjugate fluorescein- labeled anti-human IgG (goat) ready

for use.

iv. Positive control with titre information auto antibodies against granulocyte cytoplasm

(cANCA), human, ready for use.

v. Positive control with titre information auto antibodies against myeloperoxidase (anti-

MPO), human, ready for use

vi. Negative control autoantibody negative, human, ready for use.

vii. Salt for PBS pH 7.2 -

viii. Tween 20 -

ix. Embedding medium Glycerol


i. Reagent tray

ii. Test tubes and rack

iii. Pasteur and autopipettes

iv. Coplin jar

v. Distilled water

vi. Volumetric flask for PBS

vii. Wash bottles

viii. Timer

ix. Foreceps

x. Fluorescent microscope : EUROIMMUN EUROStar 3Plus Immunofluorescence

microscope with LUCAM camera model# LU375C-10.

23

2.3 Slide preparation for dsDNA

Principle:

The detection and semi-quantitation of auto antibodies aid in the diagnosis of autoimmune

diseases. With the choice of the appropriate substrate, the Kallestad® Crithidia luciliae (nDNA)

Test detects auto antibodies to native DNA (nDNA) antigens. In these procedures, a fluorescent

antibody serves as a marker for an antigen-antibody binding reaction which occurs on a substrate

surface. Among the substrates commonly used in the IFA procedure are Crithidia luciliae, a

hemoflagellate, mouse kidney and stomach, and human epithelial (HEp-2) cells. Observation of

a specific pattern of fluorescence on the substrate indicates the presence of auto antibodies in the

patient’s serum.

High titers of anti-nDNA, one type of ANA, are associated with SLE. The titer of the anti-nDNA

may decrease with successful therapy and increases in acute recurrence of the disease. Also,

DNA-anti-DNA immune complexes play a role in the pathogenesis of SLE through the deposit

of the complexes in the kidney and other tissues. For these reasons, the detection and

quantitation of anti-nDNA is diagnostically and therapeutically helpful in patients suspected or

known to have SLE.

Requirements provided with the kit :


serum+190 ul PBS)

ii. Slide Kallestad® Crithidia luciliae

iii. FITC Conjugate Fluorescein conjugated antiserum to human

Immunoglobulin.

iv. Positive control with titre information Pooled human serum with a specific autoantibody

activity

v. Negative control Pooled normal human serum.

vi. Salt for PBS pH 7.2 -

vii. Tween 20 -

viii. Evans blue counter stain -

24

2.4 Slide preparation for ASMA/AMA

Principle :

The detection and semi-quantitation of auto antibodies aid in the diagnosis of autoimmune

diseases. With the choice of the appropriate substrate, the BIORAD Kallestad Mouse

Stomach/Kidney Test detects auto antibodies to mitochondrial (AMA), smooth muscle (ASMA),

and parietal cell (APCA) antigens. In these procedures, a fluorescent antibody serves as a marker

for an antigen-antibody binding reaction which occurs on a substrate surface. Auto antibodies in

a test sample bind to antigens in the substrate. Washing removes excess serum from the

substrate. Fluorescein conjugated (FITC) antiserum added to the substrate attaches to the bound

autoantibody. After a second washing step to remove excess conjugate, the substrate is

coverslipped and viewed for fluorescent patterns with a fluorescent microscope. Observation of a

specific fluorescent pattern on the substrate indicates the presence of auto antibodies in the test

sample.

Requirements provided with the kit :

ix. Sample serum sample diluted 1:20 with PBS (10ul

serum+190 ul PBS)

x. Slide Kallestad® Mouse Stomach/Kidney, 8-well.

Mouse Stomach/Kidney

xi. FITC Conjugate Fluorescein conjugated antiserum to human

Immunoglobulin.

xii. Positive control with titre information Pooled human serum with a specific autoantibody

activity

xiii. Negative control Pooled normal human serum.

xiv. Salt for PBS pH 7.2

xv. Tween 20

xvi. Evans blue counter stain


i. Humidity chamber

ii. Glass cover slips

iii. Test tubes

iv. Pasteur and automated pipettes

v. Coplin jar

vi. Distilled water

vii. Volumetric flask for PBS

viii. Wash bottles

ix. Timer

x. Fluorescent microscope : EUROIMMUN EUROStar 3Plus Immunofluorescence

microscope with LUCAM camera model# LU375C-10.

25

Fig4. BIOCHIP coated with HEp-20-10 (Human epithelial) cells and primate liver.(10 well)

Fig 5. BIOCHIP coated with granulocytes(EOH), HEp-2 cells+ granulocytes

(HCHO).(5well)

26

Fig 6. Kallestad® Mouse Stomach/Kidney, 8-well

Fig 7. Kallestad® Crithidia luciliae, 8 well

27

Procedure :

A. Pipette: Apply 30 µl of diluted sample to each reaction field of the reagent tray, avoiding air bubbles. Transfer all samples to be tested before starting the test to a reagent tray.

B. Incubate: Start reactions by fitting the BIOCHIP Slides into the corresponding recesses of the reagent tray. Ensure that each sample makes contact with its BIOCHIP and that the individual samples do not come into contact with each other. Incubate for 30 min at room temperature (+18°C to +25°C).

C. Wash: Rinse the BIOCHIP Slides with a gentle flush of PBS–Tween using a beaker and immerse them immediately afterwards in a cuvette containing PBS–Tween for 5 min. Shake with a rotary shaker if available. Wash max. 16 slides then replace PBS-Tween with new buffer.

D. Pipette: Apply 25 µl of fluorescein-labelled anti-human globulin to each reaction field of a clean reagent tray. Fill all the fields needed before continuing incubation. The labelled anti-human serum should be mixed before use.

28

E. Incubate: Remove one BIOCHIP Slide from cuvette. Within five seconds blot only the back and the sides with a lint free paper towel and immediately fit the BIOCHIP Slide into the recesses of the reagent tray. Do not dry the areas between the reaction fields on the slide. Check for correct contact between the BIOCHIPs and liquids. Then continue with the next BIOCHIP Slide. Protect the slides from direct sunlight. Incubate for 30 min at room temperature (+18°C to +25°C).

F. Wash: Fill cuvette with new PBS-Tween. Rinse the BIOCHIP Slides with a gentle flush of PBS–Tween using a beaker and place them into the cuvette filled with the new PBS-Tween for 5 min.

G. Embed: Place embedding medium onto a cover glass – drops of max. 10 µl per reaction field. Use a reagent tray. Remove one BIOCHIP Slide from PBS–Tween and dry the back, all four sides, as well as the surface around, but not between the reaction fields with a lint free paper towel. Put the BIOCHIP Slide, with the BIOCHIPs facing downwards, onto the prepared cover glass. Check immediately that the cover glass is properly fitted into the recesses of the slide. Gently correct the position if necessary.

H. Evaluate: Read the fluorescence with the microscope. General recommendation: Objective 20x (tissue sections, infected and transfected cells), 40x (cell substrates). Excitation filter: 488 nm, color separator: 510 nm, blocking filter: 520 nm. Light source: mercury vapor lamp, 100 W, EUROIMMUN LED, EUROStar Bluelight.

29

Fig 7. EUROIMMUN EUROStar 3Plus Immunofluorescence microscope with

LUCAM camera model# LU375C-10.

30

ANA EIA as an alternative method

ANA is conventionally detected using IIF, but ELISA assays that contain a mixture of known

anti-nuclear antigens are increasingly being adopted as they are easily automated and do not

require as high a level of operator skill to perform. In the ELISA assay, ANAs are not detected

that are directed against antigens which are not included on the ELISA plate. These may include

ribosomal P and PM/Scl. It is not known whether the inability to detect antibodies to these

antigens is a clinical drawback to the use of ELISA assays. [21]

Principle of the test

Membrane strips coated with thin parallel lines of several purified, biochemically

characterised antigens are used as solid phase.The membranes are fixed as onto synthetic

foil.If the sample is positive, specific antibodies in the diluted serum sample attach to the

antigens coupled to the solid phase.

In a second incubation step, the attached antibodies react with alkaline-phosphatase-

labelled anti-human antibodies.

In a third step, the bound antibodies are stained with a chromogen/substrate solution

which is capable of promoting a color reaction. An intense dark band at the line of the

corresponding antigen appears if the serum sample contains specific antibodies.

Depending on the spectrum of antigens used, it is possible to analyse several antibodies

next to each other and simultaneously under identical conditions

.

31

RESULTS AND DISCUSSION

The 50 individuals included in the study were segregated according to the autoimmune markers

they were detected for. Among them 31 were found to be ANA +, 8 were found to be ANCA +,

and 12 were found to be positive for autoimmune liver disease (ASMA, AMA, APCA). The

primary dilution , intensity, pattern in case of ANA and the end point titer of the samples were

evaluated. The clinical history of the patients was obtained from the medical records department

of KDAH.

The main topic of discussion for this study is the mechanism of production or the reason for the

presence of auto antibodies in various diseases. These may not necessarily be autoimmune

diseases, but the presence of autoimmune markers in the serum makes for a good research topic.

The presence of an autoantibody in a patient does not assure a diagnosis of an autoimmune

disease. Rather, a positive serologic test in the company of appropriate signs and symptoms

helps to support a diagnosis. Serologic testing is flawed by the presence of autoantibodies in

healthy individuals and other patients with non-autoimmune diseases and imperfect testing

systems. Historically, many different methods were used to test for the presence an

autoantibody.

32

CASE STUDY 1: ANA IN BRAIN INJURY

2 individuals positive for ANA were found to be suffering from brain injury

A 57 year old male suffering from Diffused Cerebral Atrophy.

Other clinical findings showed rigidity of all limbs and a case history of neck injury.

Microbiological findings suspected the presence of the bacteria Streptococus Viridans

ANA blot was found to be negative.

61 year old female was diagnosed with Hypertrophic pachymeningitis

Showed symptoms of brain swelling, headache and otitis media, deafness, eye problem

Pneumatic fever.

Also tested positive for pANCA.

Both individuals showed normal biochemical and hematological reports.

Fig 9. Fluorescence pattern ANA positive Intensity : 1+ Pattern: homogenous Clinical findings : cerebral atrophy

Fig 10 Fluorescence pattern ANA positive Intensity : 2+ Pattern: cytoplasmic coarse granular. Clinical findings : Brain swelling

33

Studies suggest that immunologic tolerance against autoantigens is induced only for dominant

epitopes of such antigens. However, some kind of autoreactivity against cryptic determinants that

are generated at very low concentrations and that do not reach the threshold for recognition by

the lymphoid cells is possible. In this case the cryptic determinant might be the bacterial

commensal Streptococcus Viridans which was detected during microbiological analysis of the

individual. Recently, the hypothesis that an autoimmune disease may arise if those cryptic

determinants become visible to the immune system has gained importance. One of the

mechanisms that may unmask such determinants is apoptosis, contributing in this way to the

triggering and propagation of autoimmune diseases. In this regard, autoantigens characteristic of

systemic lupus erythematosus, which are physiologically restricted to non-cellular or nuclear

compartments, redistribute in the apoptotic cells and concentrate in the apoptotic bodies, which

explains the acute cerebral atrophy observed in the individual. However, the lipid bilayer flips

out during early apoptosis, and autoantigens that are normally in the inner membrane relocate to

the outer membrane. Both types of autoantigens can be presented to the immune cells and

generate auto antibodies.[3]

34

CASE STUDY 2: MIXED CONNECTIVE TISSUE DISORDERS

4 individuals were diagnosed with Mixed Connective Tissue Disorder i.e; Scleroderma and

Systemic Lupus Erythomatosus.

62 year old female with scleroderma.

Histopathological reports showed Nephrotic syndrome

Renal dysfunction along with microalbuminuria

33 year old female suffering from SLE

Histopathology results showed Acute renal disease and Lupus Nephritis.

Renal biopsy showed decreased C3andC4

ANA blot was positive for Anti ds DNA, Anti Histone and Nucleosome

Fig 11 Fluorescence pattern ANA positive Intensity : 3+ Pattern: centromere. Clinical findings : Scleroderma

Fig 12

Fluorescence pattern: ANA positive

Intensity : 3+

Pattern : centromere

Clinical findings : scleroderma

35

Elevated levels of ANA are found in all systemic rheumatic diseases, with sometimes high,

sometimes rather loose associations between a particular ANA specificity and a particular

rheumatic disease. Therefore, the detection and identification of ANA has gained increasing

acceptance by clinicians who use the information to help or confirm a diagnosis and in treatment

follow up. Most ANA are directed against nucleic acids or proteins associated with nucleic acids.

In Systemic Lupus Erythematosus(SLE), the most predominant antigen is probably the

nucleosome. The concept of anti‐DNA playing a direct role in the pathogenesis of SLE is based on

much circumstantial evidence. The various pieces of evidence pointing in the direction of an

active role in pathogenesis are:

1. Anti‐DNA fluctuates in time, in close association with exacerbations and remissions of the

disease: especially nephritic exacerbations are heralded by an increase in the level of

anti‐DNA, while anti‐DNA levels drop steeply during the clinical exacerbation. In fact,

upcoming exacerbations can be prevented by treatment of patients on the basis of

increasing levels of anti‐DNA .

2. Patients that do not have SLE at the time anti‐DNA is first detected in their circulation

generally develop SLE within the next 5 yr.

3. Antibodies to DNA can be eluted from affected kidneys.

4. DNA and antibodies to DNA leads to the binding of anti‐DNA to the glomerular basement

membrane. Initially thought to be based on anti‐DNA cross‐reactivity, we now know this

binding is mediated by nucleosomes.

Taken together, these pieces of evidence indicate that anti‐DNA is directly implicated in the

induction and propagation of inflammatory reactions in affected tissues. It is the current view

that autoantigens themselves drive the autoimmune response against them. In this view,

nucleosomes are probably the most relevant autoantigens for the genesis of antibodies to

nucleosomes, histones and DNA.

Fig 14. Fluorescence pattern Anti-dsDNA positive Intensity : 3+. Clinical findings : SLE

36

Fig 30. a butterfly rash typically seen in SLE

Fig 31. juvenile Rheumatoid Arthritis

Fig 13. Fluorescence pattern ANA positive Intensity : 3+ Pattern: cytoplasmic. Clinical findings : SLE

37

CASE STUDY 3: AUTOIMMUNE MARKERS IN PREGNANCY RELATED

DISORDERS

2 females suffering from pregnancy related disorders, i.e; pregnancy induced

hypertension and miscarriage.

Both the females showed signs of retrosternal discomfort.

Going through pregnancy induced hypertension.

They showed decreased hemoglobin and hematocrit count.

Had history of high blood pressure and pre-eclampsia.

All AIDs, to some extent, have implications for fertility and obstetrics. In the general population,

about 80% of miscarriages occur in the first 12 weeks of pregnancy and the risk of miscarriage in

those under the age of 35 is about 10% while it is about 45% in those over the age of 40. Most AIDs

occur frequently in women and should they appear at childbearing age, they pose a potential risk

for almost all aspects of reproduction, from fertility to pregnancy itself. In the past, it was

suggested that women with certain AIDs [particularly systemic lupus erythematosus (SLE)

Fig 16. Fluorescence pattern ANA positive Intensity : 2+ Pattern: speckled. Clinical findings : Miscarriage

Fig 15. Fluorescence pattern ANA positive Intensity : 1+ Pattern: Cytoplasmic, more S/O of JO-1 Clinical findings : pregnancy induced hypertension.

38

/antiphospholipid syndrome (APS)] should avoid pregnancy. The absence of Antinuclear

Antibody in the blood is a negative result and generally indicates that there is no autoimmune

disorder present. Significant levels of ANA in the blood can indicate the presence of an

autoimmune disorder. Lower level, but still positive results, can be obtained in a pregnant woman

without other symptoms of autoimmune disease. This does not mean that she has or will develop

a serious autoimmune reaction, but it can mean that the immune system has the potential to

develop antibodies against a developing fetus, a situation that in the absence of treatment could

result in the ending of the pregnancy. Low-level positive results in a woman who has recently

experienced a miscarriage can be interpreted to mean that an autoimmune response might have

been responsible for the ending of her pregnancy. Currently, due to available treatments and

specialised care for pregnant women with AID, the prognosis for both mother and child has

improved significantly. However, these pregnancies are always high risk, often associated with

foetal loss in the first trimester, preeclampsia/eclampsia, intrauterine growth restriction,

premature rupture of membranes, placental insufficiency, pre-term birth, caesarean delivery and

low birth weight. The overall principle, common to all AIDs, is planning the pregnancy for the

remission phase of the disease, in addition to all the care necessary for a successful pregnancy. A

variety of treatment options exist that can maximize the probability of a successful pregnancy for

women who have autoimmune issues. In the above cases treatments include the steroid

prednisone, aspirin or the drug heparin, which is a commonly used blood thinner.[8]

As shown in this and other studies, ANA reactivity is greater in women than men, although these

gender differences did not occur with antibodies to citrullinated proteins. In an era of genetics

and personalized medicine, the biological diff erences between women and men sometimes do

not get the attention they deserve. While the role of hormones compared to the genetic

endowment of two Xs versus an XY tandem can be debated, nevertheless, women appear

predisposed to lupus as well as baseline ANA reactivity. In the future, consideration of the role of

pregnancy in ANA reactivity seems worthwhile since, during normal pregnancy, there can be

extensive exposure to nuclear antigens. Furthermore, although often considered a time of

immunosuppression, pregnancy actually shows a surge of inflammatory activity that could impact

on immune responsiveness[17]

39

5 individuals were suspected for rheumatic disease i.e; Rheumatoid arthritis

Fig 17. Fluorescence pattern ANCA positive Intensity : 2+ Pattern: pANCA. Clinical findings : Rheumatoid arthritis

Fig 18. Fluorescence pattern ANA positive Intensity : 1+ Pattern: antibodies against spindle fibres… Clinical findings : Rheumatoid arthritis

40

CASE STUDY 5: AUTOIMMUE MARKERS IN ANEMIA

3 individuals were suspected of having blood related disorders i.e; hemolytic anemia,

megaloblastic anemia and acute thrombocytopenia.

All the 3 undividuals showed a declining hemoglobin count

WBC count was significantly high upto 15000

Genetics and environmental factors, such as acute viral infection, can induce transient

autoimmune responses, including the generation of auto-antibodies. In an acute DV infection,

anti-dengue NS1 antibody can cross-react with endothelial cells and antibody can inhibit the

active form of plasminogen through molecular mimicry.[10] Binding of antibodies to RBCs

activates the classical pathway of the complement system leading to the formation of membrane

attack complex and intravascular haemolysis.On the other hand, if classic pathway is ineffective,

RBCs are opsonised with complement proteins ( particularly C3b and C4b) which enhances

phagocytosis in liver and spleen, presenting as extravascular haemolysis.[23]

Fig 19. Fluorescence pattern ANA positive Intensity : 3+ Pattern: nucleolar. Clinical findings : Acute thrombocytopenia.

Fig 20. Fluorescence pattern ANA positive Intensity : 1+ Pattern: cytoplasmic. Clinical findings : Megaloblastic anaemia.

41

CASE STUDY 6: AUTOIMMUNE MARKERS IN HEALTHY INDIVIDUALS

8 individuals were found to be healthy

The use of autoantibodies, one of the most venerable tests in immunology, while potentially very

informative, faces major challenges. Amongst these, the frequency of serological positivity in the

general population is probably the greatest. While the actual frequency of positive assays varies

with methodology, nevertheless, up to 20% or more of otherwise healthy people can express an

ANA. The expression of these antibodies does not appear related to age despite ideas that

immunosenescence may promote autoreactivty.

The basis of this seropositivity is puzzling. One possibility is that ANA reactivity represents

vagaries of the assays, allowing detection of antibodies of either low titer or low avidity. Many

nuclear antigens are highly charged molecules, with DNA and histones the prime examples. As

such, ANA binding may occur by charge-charge interactions or cross-reactivity with other

antigens (also charged). In this regard, solid phase or multiplex assays may reveal a different

Fig 21. Fluorescence pattern ANA positive Intensity : 1+ Pattern: few nucleolar dots. Clinical findings : healthy individual

Fig 22 Fluorescence pattern ANA positive Intensity : 1+ Pattern: cytoplasmic Clinical findings : healthy individual

42

perspective on serology than the classic (and now antiquated) methods. These older assays

required large amounts of antibody for detection, such as the formation of precipitating

complexes in immunodiffusion assays. As a result, seropositivity indicated a robust response.

While the solid phase and multiplex assays are sensitive and allow high throughput, their

interpretation requires caution, especially in the setting of preclinical or subclinical disease,

where the measured responses may be low.

Another explanation for the frequency of ANA expression in the general population relates to

intrinsic immunological disturbances among humans. Perhaps as a species, humans are

predisposed to autoimmunity, with ANA expression the tip of the iceberg of autoimmunity.[17]

Fig 23. Fluorescence pattern ASMA positive Intensity : 1+ Status: healthy individual

43

8 individuals were suffering from ANCA asociated vasculitis

3 patients suffered from chronic kidney diseases.

2 patients showed glomerulonephritis.

2 patients depicted pulmonary disorders.

1 individual was diagnosed with small vessel vasculitides

Anti-neutrophil cytoplasmic antibodies (ANCA) have become important diagnostic markers of

small vessel vasculitides characterized by focal necrotizing lesions of vessel walls and

accumulation of lymphocytes and macrophages around the affected vessels. IgG class ANCA

directed to proteinase 3 (PR3) of neutrophils and monocytes seem to be directly involved in the

pathophysiology of vascular damage by causing excessive neutrophil activation and vessel wall

destruction. PR3 and elastase are important players in the mechanisms of vascular necrosis.

Fig 24 Fluorescence pattern ANCA positive Intensity : 1+ Pattern: cANCA Status: SLE, acute renal disease

Fig 25 Fluorescence pattern ANCA positive Intensity : 1+ Pattern: pANCA Clinical findings : Rheumatoid arthritis

44

Methods of detecting ANCA have now been defined but are not uniformly used, even though

clinical decisions heavily depend on correct ANCA results.

It is now well recognized that C-ANCA/PR3-ANCA is found in about 80% of patients with

Wegener’s granulomatosis, and also in about 35% of patients with microscopic polyangiitis,

Churg–Strauss syndrome, and renal-limited rapidly progressive glomerulonephritis. The

respective frequencies of P-ANCA/MPO-ANCA in these diseases are around 15%, 50%, 40%, and

50%. The prevalence of PR3-ANCA in patients with Wegener’s granulomatosis depends greatly on

the vasculitic disease activity and the extent of vasculitis when the blood is sampled. A practical

consequence of finding positive CANCA/ PR3-ANCA results has been a great increase in the

correct diagnosis of small-vessel vasculitides. Preliminary data indicate that fluctuations in PR3-

ANCA levels as measured by capture EIA may better reflect disease activity than direct EIA

methods. A number of clinical differences have been found between vasculitis in patients with

PR3-ANCA and those with MPO-ANCA. The former have more upper-airway involvement and are

more likely to get clinical relapses of their disease than MPO-ANCA-positive patients, who exhibit

more renopulmonary involvement and get fewer relapses. Granulomatous lesions are more

common in patients with PR3-ANCA than in those with MPO-ANCA.

45

11 individuals were found to be suffering from Liver diseases

These are the different disorders found in the patients:-

5 patients suffering from Chronic alcoholic liver disease

These patients showed signs of enlarged liver and spleen.

23 year old male died of fulminant hepatic failure. He was detected with Viral

Hepatitis E

2 individuals suffering from Autoimmune Hepatitis

2 individuals sufferin from Primary billiary cirrhosis

33 year old female suffering from Wilson’s disease.

The classification of Auto Immune Hepatitis into different types is based on serum autoantibody

profiles. Type I AIH is characterized by the presence of antinuclear antibody (ANA), anti smooth

muscle antibody (SMA), or both and constitutes 80% of AIH cases. About 25% have cirrhosis at

presentation, and association with other autoimmune diseases is common. Although the exact

etiopathogenesis is unknown, AIH, like many autoimmune diseases, is thought to be caused by

environmental triggers and failure of immune tolerance mechanisms in a genetically susceptible

host. These triggers may be of viral or drug etiology, but most cases have an unknown trigger.

Triggers may share epitopes that resemble self-antigens, and molecular mimicry between foreign

antigens and self-antigens is the most frequently proposed initiating mechanism in type 2 AIH

where the autoantigen is known. Repeated exposures to the triggering antigen, in turn, may

trigger autoreactive organ-specific responses. The characteristic circulating auto antibodies seen

in AIH include ANA, SMA, and (LKM-1) auto antibodies. They are helpful in diagnosis as well as

for classification of AIH into type 1 and type 2 diseases

Fig 26 Fluorescence pattern ASMA positive Intensity : 2+ Clinical findings : Autoimmune Hepatitis

46

Primary biliary cirrhosis (PBC) is a chronic progressive cholestatic liver disease characterized by

immune-mediated destruction of small- and medium-sized intrahepatic bile ducts and the

presence of antimitochondrial antibodies (AMA) in serum of affected patients. PBC is

considered a model autoimmune disease on the basis of several features, including the presence

of a highly direct and very specific immune response to mitochondrial autoantigens, female

predominance, and homogeneity among patients. The immunopathology of PBC is characterized

by the presence of CD4+ and CD8

+ T-cell infiltrates in the liver and targeted destruction of

biliary epithelial cells (BECs). This suggests that BECs may have unique immunological

characteristics.

Figure 32: Interface hepatitis demonstrated by infiltration of lymphoplasmacytic infiltrate

into the hepatic parenchyma typical of autoimmune hepatitis.[7]

Fig 27. Fluorescence pattern ASMA positive Intensity : 2+ Clinical findings : Fulminant hepatic failure. Viral Hepatitis E

47

While it is currently accepted that AMA are the most specific serological markers of PBC, more

than 60 auto antibodies have been investigated in PBC patients, some having previously been

considered specific for other autoimmune diseases. AMA are detected in over 90% of patients

with PBC, whereas their prevalence in the general population is extremely low, varying between

0.16% and 1%, and only reaching 8% in hepatitis C virus (HCV)-infected patients. AMA

seropositivity is a strong predictor for the development of PBC. The AMA target antigens are all

localized within the inner mitochondrial matrix and catalyze the oxidative decarboxylation of 2-

oxo-acid acid substrates. In approximately 95% of patients, AMA are directed towards the 74 kD

mitochondrial polypeptide identified as PDC-E2. During apoptosis of BECs, PDC-E2 remains

immunologically intact without being glutathiolated, and becomes the source of the PDC-E2

apotope. The term apotope specifies an epitope created during the processes of apoptosis. PDC-

E2 contained within apoptotic bodies can be recognized by circulating AMA, and the resulting

apotope-AMA complex then stimulates the innate immune systems in genetically susceptible

individuals

Fig 28. Fluorescence pattern AMA positive Intensity : 3+ Clinical findings : Primary billiary cirrhosis

Fig 29. Fluorescence pattern APCA positive Intensity : 3+ Clinical findings : Polymyositis

48

CONCLUSION

Study of autoimmune markers was carried out by Indirect Immunofluoresence Assay. The

markers included in the study are ANA, ANCA, Anti-dsDNA, and ASMA. The role and

importance of these markers in autoimmune diseases can be described as follows :-

i. Autoantibodies as markers to define and classify disease

In patients with established disease, autoantibodies can help define the nature of the disease and

provide markers to classify the disease. For example, type 1 diabetes, thyroiditis, and adrenalitis

are classified as autoimmune or not autoimmune, based on the presence or absence of disease-

associated antibodies. Similarly, there are several causes of atrophic gastritis and of vitamin

B12 deficiency, but the combination of the two, in association with autoantibodies to parietal

cells or intrinsic factor, indicates that the cause is autoimmune gastritis, also called pernicious

anemia (PA).

ii. Autoantibodies as markers to predict disease

Since autoantibodies are markers of disease activity, it follows that, at least under some

circumstances, autoantibodies should be able to predict disease. This approach is especially

promising for diseases with a long preclinical period, a feature of many organ-specific

autoimmune diseases.

Three parameters must be carefully quantitated for predictive tests to be clinically useful:

sensitivity of prediction, specificity of prediction, and positive predictive values.[24]

49

CURRENT STATE AND FUTURE

PROGRESS

As our understanding of the molecular and cellular aspects of autoimmunity increases, we will

continue to see more effective treatments for these diseases. For example, multiple sclerosis

(MS) is an autoimmune neurological disorder thought to be mediated by antigen-specific CD4+

T helper (Th1) T cells which cause demyelination of the central nervous system (CNS). Many

current therapeutic strategies attempt to downregulate the entire immune system by causing

generalized immunosuppression, in the hope that this will reduce the specific action of the T

cells involved. This approach, incidentally, is widespread in autoimmune treatments in the

absence of better options. Unfortunately, generalized immunosuppression has not met with the

success expected. Now, new approaches tend to employ therapies based on immunomodulation,

rather than immunosuppression, by administration of cytokines such as interferon (IFN)-β and

glatiramer acetate, and in the case of MS, these approaches are proving to be more effective.

Refinements in our understanding of the effects of immunomodulation versus more drastic

measures will no doubt help us devise even more effective therapies.

Finally we can say that, Autoimmune disorders are a family of diseases that represent a major

societal burden. While the pace of our understanding of the molecular and cellular processes of

these complex disorders have thus far been behind that of other diseases, the advent of largescale

genomic and functional analysis tools are now helping redress the balance. The enormous

pressure to understand these diseases and cure them is the best guarantee of progress in this

area.[11]

50

REFERENCES 1. Antinuclear antibodies and their detection methods in diagnosis of connective tissue

diseases: a journey revisited Yashwant Kumar*1, Alka Bhatia2 and Ranjana Walker

Minz3

2. Antinuclear antibodies and their detection methods in diagnosis of connective tissue

diseases: a journey revisited

3. ARTHRITIS & RHEUMATISM

Vol. 46, No. 12, December 2002, pp 3290–3300

DOI 10.1002/art.10684© 2002, American College of Rheumatology

Induction of Cell Death by Sera From Patients With Acute

Brain Injury as a Mechanism of Production of Autoantibodies

Herminio Lo´pez-Escribano,1 Eduardo Min˜ambres,1 Moise´s Labrador,2 Marı´a Jose

4. NIH Public Access

Author Manuscript

J Allergy Clin Immunol. Author manuscript; available in PMC 2011 February 1.

Published in final edited form as: J Allergy Clin Immunol. 2010 February ; 125(2 Suppl 2):

Diagnostic Testing and Interpretation of Tests for Autoimmunity Christine Castro,

D.O. and Mark Gourley, M.D.

51

National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National

Institutes of Health (NIH), Bethesda, MD, U.S.A

5. Open Journal of Internal Medicine, 2014, 4, 59-63

Published Online September 2014 in SciRes. http://www.scirp.org/journal/ojim

http://dx.doi.org/10.4236/ojim.2014.43009

Medicine Service. Open Journal of

Internal Medicine, 4, 59-63. http://dx.doi.org/10.4236/ojim.2014.43009

Pernicious Anemia Associated Autoimmune

Diseases in a Sub Saharian African Internal

Medicine Service

Abdoulaye Pouye*, Seynabou Fall Dieng, Daher Abdoukarim Oumar,

Fatou Samba Diago N’Diaye, Nafissatou Diagne Sakho, Atoumane Faye,

Souhaibou N’Dongo

Medical Clinic 1 University Teaching Hospital Aristide le Dantec/Cheikh Anta Diop

University of Dakar,

Dakar, Senegal

Email: *[email protected]

Received 27 June 2014; revised 24 July 2014; accepted 20 August 2014

Copyright © 2014 by authors and Scientific Research Publishing Inc.

6. Study of anti nuclear and anti smooth muscle antibodies

in patients with chronic obstructive pulmonary disease

Tahany M. Gouda a, Ayman A. Yousof a, Mahmoud M. Al Salahy a,

Gehan F. Al mehy a, Tarek S. Essawy a,*, Osama S. El-Shaer b

a Department of Chest Diseases, Benha University, Egypt

b Department of Clinical and Chemical Pathology, Benha University, Egypt

Received 8 October 2013; accepted 6 November 2013

Available online 13 December 2013

7. AutoimmuneHepatitis: A Review of Current

Diagnosis and Treatment

AshimaMakol,1 Kymberly D.Watt,2 and Vaidehi R. Chowdhary1

1Division of Rheumatology, Department of Medicine, Mayo Clinic College of Medicine,

Rochester, MN 55905, USA

2Division of Gastroenterology and Hepatology, Department of Medicine,Mayo Clinic

College of Medicine,

Rochester, MN 55905, USA

Received 13 November 2010; Revised 15 February 2011; Accepted 3 March 2011

Academic Editor: Mikio Zeniya

8. Autoimmune diseases and pregnancy: analysis of a series of cases

Vânia Gomes1, Alexandra Mesquita2 and Carlos Capela1,2*

9. Significance of smooth muscle/anti-actin autoantibodies in celiac disease

Silvia Pedreira,1 Emilia Sugai,1 María Laura Moreno,1 Horacio Vázquez,1

52

Sonia Niveloni,1 Edgardo Smecuol,1 Roberto Mazure,1 Zulema Kogan,2

Eduardo Mauriño,1,3 Julio C Bai 1,3,4

Acta Gastroenterol Latinoam 2005;35:83-93

10. International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Long-term persistence of clinical symptoms in dengue-infected persons

and its association with immunological disorders Gissel Garcıá a,*, Narjara Gonza´ lez b, Ana Beatriz Pe´ rez a, Beatriz Sierra a, Eglis

Aguirre a,

Damaris Rizo c, Alienys Izquierdo a, Lizet Sańchez a, Danay Dıáz a, Magnolia Lezcay a,

Betsi Pacheco a, Kenji Hirayama d, Maria G. Guzmań a

11. © 2000 Nature America Inc. • http://biotech.nature.com

Autoimmune disease

Rapid progress in our understanding of immune function promises

more effective treatments for autoimmune disorders.

12. Meta-Analysis: Diagnostic Accuracy of Antinuclear Antibodies, Smooth Muscle

Antibodies and Antibodies to a Soluble Liver Antigen/Liver Pancreas in

Autoimmune Hepatitis

Wen-Chao Zhang1., Feng-Rong Zhao2., Juan Chen3, Wei-Xian Chen1*

1 The Department of Laboratory Medicine, the Second Affiliated Hospital of Chongqing

Medical University, Chongqing, China, 2 The Department of Gynaecology and

Obstetrics, Youyang People’s Hospital, Chongqing, China, 3 Institute of Viral Hepatitis, the

Second Affiliated Hospital of Chongqing Medical University, Chongqing, China

13. What you should know about PR3-ANCA :An introduction

Allan Wiik Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark

14. BSR and BHPR guideline for the management of adults with ANCA-associated

vasculitis

Eleana Ntatsaki1,2, David Carruthers3, Kuntal Chakravarty4, David D’Cruz5,

Lorraine Harper6, David Jayne7, Raashid Luqmani8, John Mills9,

Janice Mooney10, Michael Venning11 and Richard A. Watts12,13, on behalf of the

BSR and BHPR Standards, Guidelines and Audit Working Group

15. EUROIMMUN ANA IFA: HEp-20-10 Test instruction manual

www.euroimmun.us

16. Antinuclear antibodies in healthy people: the tip of autoimmunity’s iceberg? David S Pisetsky1,2

See related research by Li et al., http://arthritis-research.com/content/13/2/R38EDITORIAL *Correspondence: [email protected]

1Medical Research Service, Durham Veterans Administration Medical Center,

508 Fulton St, Durham, NC 27705, USA

Full list of author information is available at the end of the article

http://www.elsevier.com/locate/ijid

http://biotech.nature.com/

http://www.euroimmun.us/

53

17. Autoantibodies in Autoimmune Pancreatitis

Daniel S. Smyk,1 Eirini I. Rigopoulou,2 Andreas L. Koutsoumpas,1 Stephen Kriese,1

Andrew K. Burroughs,3 and Dimitrios P. Bogdanos1, 2, 4

1 Institute of Liver Studies, King’s College London School of Medicine, King’s College

Hospital, Denmark Hill,

London SE5 9RS, UK

2Department of Medicine, University of Thessaly Medical School, Viopolis, 41110 Larissa,

Greece

3 The Sheila Sherlock Liver Centre and University Department of Surgery, Royal Free

Hospital, London NW3 2QG, UK

4Research Group of Cell Immunotherapy and Molecular Immunodiagnostics, Institute of

Biomedical Research & Technology,

Centre for Research and Technology-Thessaly (CE.RE.TE.TH), 41222 Larissa, Greece

18. Autoantibodies in primary biliary cirrhosis: Recent progress in research on the

pathogenetic and clinical significance

Satoshi Yamagiwa, Hiroteru Kamimura, Masaaki Takamura, Yutaka Aoyagi

World J Gastroenterol 2014 March 14; 20(10): 2606-2612

ISSN 1007-9327 (print) ISSN 2219-2840 (online)

© 2014 Baishideng Publishing Group Co., Limited. All rights reserved.

19. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme

Immunoassays and Immunofluorescence Assays

Renata Baronaite,1,2 Merete Engelhart,2 Troels Mørk Hansen,2 Gorm Thamsborg,3

Hanne Slott Jensen,2 Steen Stender,1 and Pal Bela Szecsi1

1 Department of Clinical Biochemistry, Gentofte Hospital, University of Copenhagen, 2900

Hellerup, Denmark

2Department of Rheumatology, Gentofte Hospital, University of Copenhagen, 2900

Hellerup, Denmark

3Department of Rheumatology, Glostrup Hospital, University of Copenhagen, 2600

Glostrup, Denmark

20. ANA measured by ELISA

GERALD A. MAGUIRE 1, AMEL GINAWI 2, JEFFREY LEE, ANITA Y. N. LIM,

GRAHAM WOOD, SALLY HOUGHTON, DINAKANTHA S. KUMARARATNE, HILL J.

S. GASTON

1Department of Clinical Biochemistry and Immunology,

2Department of Rheumatology, Addenbrooke’s Hospital and

3Division of Rheumatology, Department of Medicine, University

of Cambridge, Cambridge, UK

Accepted 22 April 2009

21. Anti-dsDNA Antibodies are one of the many autoantibodies in

systemic lupus erythematosus [version 1; referees: 2 approved]

Shu Man Fu , Chao Dai , Zhenhuan Zhao , Felicia Gaskin4

54

Division of Rheumatology, University of Virginia, Box 800133, Charlottesville, VA, 22908-

0133, USA

Center for Immunity, Inflammation and Regenerative Medicine, Department of Medicine,

University of Virginia, Box 800133, Charlottesville,

VA, 22908-0133, USA

Department of Microbiology, Immunology and Cancer Biology, School of Medicine,

University of Virginia, Charlottesville, VA, 22908, USA

Department of Psychiatry and Neurobehavioral Sciences, School of Medicine, University of

Virginia, Charlottesville, VA, 22908, USA

22. Cold agglutinin-induced haemolysis in association with antinuclear antibody-

negative SLE

Vinod K Chaubey, Lovely Chhabra

Department of Internal

Medicine, Saint Vincent

Hospital, University of Massachusetts Medical School,

Worcester, Massachusetts, USA

23. Autoantibodies as predictors of disease

David Leslie,1 Peter Lipsky,2 and Abner Louis Notkins3

1Department of Diabetes and Metabolism, St. Bartholomew’s Hospital, London, United

Kingdom

2Laboratory of Autoimmunity, National Institute of Arthritis and Musculoskeletal and Skin

Diseases, and

3Experimental Medicine Section, Oral Infection and Immunity Branch, National Institute of

Dental and Craniofacial Research,

NIH, Bethesda, Maryland, USA

Address correspondence to: David Leslie, Department of Diabetes and Metabolism, St.

Bartholomew’s Hospital, London EC1A 7BE,

United Kingdom. Phone: 44-0-207-601-7450; Fax: 44-0-207-601-7449; E-mail:

[email protected].

J. Clin. Invest. 108:1417–1422 (2001). DOI:10.1172/JCI200114452.

24. PROGRESS IN AUTOIMMUNE DISEASES RESEARCH

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of

Health

National Institute of Allergy and Infectious Diseases

25. Serum Autoantibodies: From Identification to Clinical Relevance

Pietro Invernizzi,1 Xavier Bossuyt,2 and Dimitrios P. Bogdanos3,4,5

1 Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research

Center,

Via Manzoni 56, 20089 Rozzano, Italy

2 Laboratory Medicine, University Hospitals Leuven and Experimental Laboratory

Immunology, Department of Microbiology

and Immunology, KU Leuven. Herestraat 49, 3000 Leuven, Belgium

55

3Department of Medicine, Faculty of Medicine, School of Health Sciences, University

ofThessaly,

Biopolis, 41110 Larissa, Greece

4Cellular Immunotherapy and Molecular Immunodiagnostics, Biomedical Section, Institute

of Research and Technology Thessaly,

41222 Larissa, Greece

5 Institute of Liver Studies, King’s College London School of Medicine King’s College

Hospital, Denmark Hill Campus,

London SE5 9RS, UK

autoimmune markers thesis

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