1. M.Sc part 2
Seat no. 40231
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,
MD (Biochemistry), DNB, PGDHHM, Green Belt (Six Sigma)
Consultant, Biochemistry & Immunology,
Kokilaben Dhirubhai Ambani Hospital & Medical Research
Institute, Mumbai.

2. AUTOIMMUNE DISEASE
 An autoimmune disease occurs when the body's
immune system begins to attack its own antigens.
 In general, these diseases are associated with
humoral or cell-mediated immune reactions
against one or more of the body’s own
constituents

3. CLASSIFICATION
AUTOIMMUNE DISEASEAUTOIMMUNE DISEASE
SYSTEMICSYSTEMIC
SLE
SCLERODERMA
RHEUMATOID ARTHRITIS
SJOGREN’S SYNDROME
MCTD
POLYMYOSITIS /DERMATOMYOSITIS
ORGAN SPECIFICORGAN SPECIFIC
THYROIDITIS
DIABETES MELLITUS
BILLIARY CIRRHOSIS

4. AUTOIMMUNE MARKERS
 The markers used 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

5. Indirect immunofluorescence
HEp-2 CELLS
FITC TAGGED ANTI
HUMAN ANTIBODY
Fluorescent microscopy

6. SLIDE PREPARATION

7. .

8. .
 BIOCHIP coated with HEp-20-10 (Human epithelial) cells and
primate liver.(10 well)
 BIOCHIP coated with granulocytes(EOH), HEp-2 cells+
granulocytes (HCHO).(5well)

9. .
Kallestad® Mouse Stomach/Kidney, 8-well
Kallestad® Crithidia luciliae, 8 well

10. OBSERVATIONS
 ANA patterns
 Homogenous

11. .
 Speckled
 Cytoplasmic

12. .
 Nucleolar
 Spindle fibre

13. ANCA patterns
pANCA
cANCA

14. Ds-DNA pattern

15. AILD marker patterns
 ASMA

16. .
 AMA
 APCA

17. 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.

18. CASE STUDY 1: ANA IN BRAIN
INJURY
 Diffused Cerebral Atrophy.
 Hypertrophic pachymeningitis
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.
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.

19. CASE STUDY 2: MIXED
CONNECTIVE TISSUE DISORDERS
 Scleroderma .
 Systemic Lupus Erythomatosus.
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.

20. CASE STUDY 3: AUTOIMMUNE MARKERS
IN PREGNANCY RELATED DISORDERS
 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.
 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.

21. CASE STUDY 5: AUTOIMMUE
MARKERS IN ANEMIA
 Hemolytic anemia
 Megaloblastic anemia
 Acute thrombocytopenia due to dengue
Genetics and environmental factors, such as acute viral infection, can induce
transient autoimmune responses, including the generation of auto-
antibodies. In an acute infection, anti-dengue NS1 antibody can cross-react
with endothelial cells and antibody can inhibit the active form of
plasminogen through molecular mimicry. 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.

22. CASE STUDY 6: ANCA IN ANCA
ASSOCIATED VASCULITIS
 Chronic kidney diseases.
 Glomerulonephritis.
 Pulmonary disorders.
 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.

23. CASE STUDY 7: AUTOIMMUNE
MARKERS IN LIVER DISEASES
 Chronic alcoholic liver disease
 Viral Hepatitis E
 Autoimmune Hepatitis
 Primary billiary cirrhosis
 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.
Although the exact etiopathogenesis is unknown, it 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

24. CASE STUDY 8: AUTOIMMUNE
MARKERS IN HEALTHY
INDIVIDUALS 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.
 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).
 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

25. CONCLUSION
 i. Autoantibodies as markers to define and classify 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.

26. 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 larges cale 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.

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