1. A Study of
Autoimmune
markers by Indirect
Immunofluoresence
Assay :
ANA , ASMA , ANCA and dsDNA
.
2016
Moiz Indorewala
[Type the company name]
3/23/2016
2. 2
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
3. 3
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. 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.
5. 5
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.
6. 6
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
7. 7
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 12
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
10 Fluorescence pattern ANA positive 26
11 Fluorescence pattern ANA positive 28
12 Fluorescence pattern ANA positive 29
13 Fluorescence pattern ANA positive 30
14 Fluorescence pattern Anti-dsDNA positive 32
15 Fluorescence pattern ANA positive 36
8. 8
16 Fluorescence pattern ANA positive 39
17 Fluorescence pattern ANCA positive 39
18 Fluorescence pattern ANA positive 40
19 Fluorescence pattern ANA positive 48
20 Fluorescence pattern ANA positive 51
21 Fluorescence pattern ANA positive 53
22 Fluorescence pattern ANA positive 53
23 Fluorescence pattern ASMA positive 54
24 Fluorescence pattern ANCA positive 57
25 Fluorescence pattern ANCA positive 58
26 Fluorescence pattern ASMA positive 59
27 Fluorescence pattern ASMA positive 74
28 Fluorescence pattern AMA positive 75
29 Fluorescence pattern APCA positive
30 A butterfly rash typically seen in SLE
31 Juvenile Rheumatoid Arthritis
9. 9
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.
10. 10
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
11. 11
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)
12. 12
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
13. 13
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)
14. 14
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
15. 15
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]
16. 16
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]
17. 17
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.
18. 18
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
19. 19
. 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.
20. 20
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.
21. 21
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. 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:
i. Sample serum sample diluted 1:10 with PBS (10ul
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
Miscellaneous requirements:
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. 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 :
i. Sample serum sample diluted 1:10 with PBS (10ul
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. 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
Miscellaneous requirements:
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. 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)
27. 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. 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. 29
Fig 7. EUROIMMUN EUROStar 3Plus Immunofluorescence microscope with
LUCAM camera model# LU375C-10.
30. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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]
54. 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:
r.d.g.leslie@mds.qmw.ac.uk.
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. 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
![A Study of
Autoimmune
markers by Indirect
Immunofluoresence
Assay :
ANA , ASMA , ANCA and dsDNA
.
2016
Moiz Indorewala
[Type the company name]
3/23/2016](https://image.slidesharecdn.com/39965421-e9bb-4faa-b8e6-50a7e0df38a7-161227161227/85/autoimmune-markers-thesis-1-320.jpg)
