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Detection of the patterns of ANA by immunofluorescence in autoimmune diseases

This study describes the various autoimmune diseases and the patterns of ANA by iimmunofluorescent study. download free pdf
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Published Date:31-07-2017
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D DE ET TE EC CT TI IO ON N A AN ND D C CH HA AR RA AC CT TE ER RI IZ ZA AT TI IO ON N O OF F P PA AT TT TE ER RN NS S O OF F A AN NT TI IN NU UC CL LE EA AR R A AN NT TI IB BO OD DI IE ES S B BY Y I IN ND DI IR RE EC CT T I IM MM MU UN NO OF FL LU UO OR RE ES SC CE EN NC CE E I IN N A AU UT TO OI IM MM MU UN NE E D DI IS SE EA AS SE ES S B By y Dr. VIDHYAVATHI.V Dr. VIDHYAVATHI.V D Diis ss se er rtta attiio on n S Su ub bm miitttte ed d tto o tth he e R Ra ajjiiv v G Ga an nd dh hii U Un niiv ve er rs siitty y o of f H He ea alltth h S Sc ciie en nc ce es s,, B Ba an ng ga allo or re e,, Karnataka in partial fulfillment of the requirements for the degree of Karnataka in partial fulfillment of the requirements for the degree of M M.. D D.. ( (P PA AT TH HO OL LO OG GY Y) ) Under the guidance of Under the guidance of D Dr r.. G GE EE ET TH HA AM MA AN NI I V V.. M M.. D D.. Professor and Head Professor and Head D DE EP PA AR RT TM ME EN NT T O OF F P PA AT TH HO OL LO OG GY Y K KE EM MP PE EG GO OW WD DA A I IN NS ST TI IT TU UT TE E O OF F M ME ED DI IC CA AL L S SC CI IE EN NC CE ES S B BA AN NG GA AL LO OR RE E 2011 2011 i R RA AJ JI IV V G GA AN ND DH HI I U UN NI IV VE ER RS SI IT TY Y O OF F H HE EA AL LT TH H S SC CI IE EN NC CE ES S D DE EC CL LA AR RA AT TI IO ON N B BY Y T TH HE E C CA AN ND DI ID DA AT TE E I I h he er re eb by y d de ec clla ar re e tth ha att tth hiis s d diis ss se er rtta attiio on n ttiittlle ed d “ “D DE ET TE EC CT TI IO ON N A AN ND D C CH HA AR RA AC CT TE ER RI IZ ZA AT TI IO ON N O OF F P PA AT TT TE ER RN NS S O OF F A AN NT TI IN NU UC CL LE EA AR R A AN NT TI IB BO OD DI IE ES S BY INDIRECT IMMUNOFLUORESCENCE IN AUTOIMMUNE DISEASES ” BY INDIRECT IMMUNOFLUORESCENCE IN AUTOIMMUNE DISEASES ” is a bonafide and genuine research work carried out by me under the guidance of is a bonafide and genuine research work carried out by me under the guidance of Dr. GEETHAMANI V. MD, Professor and Head, Department of Pathology, Dr. GEETHAMANI V. MD, Professor and Head, Department of Pathology, K Ke em mp pe eg go ow wd da a I In ns sttiittu utte e o of f M Me ed diic ca all S Sc ciie en nc ce es s,, B Ba an ng ga allo or re e,, iin n p pa ar rttiia all f fu ullf fiillllm me en ntt o of f tth he e regulations of Rajiv Gandhi University of Health Sciences for the award of M. D. regulations of Rajiv Gandhi University of Health Sciences for the award of M. D. D De eg gr re ee e iin n P Pa atth ho ollo og gy y.. D Da at te e: : S Siig gn na at tu ur re e o of f t th he e C Ca an nd diid da at te e Place: Name: Dr. VIDHYAVATHI.V Place: Name: Dr. VIDHYAVATHI.V ii C CE ER RT TI IF FI IC CA AT TE E B BY Y T TH HE E G GU UI ID DE E T Th hiis s iis s tto o c ce er rttiif fy y tth ha att tth hiis s d diis ss se er rtta attiio on n ttiittlle ed d “ “D DE ET TE EC CT TI IO ON N A AN ND D CHARACTERIZATION OF PATTERNS OF ANTINUCLEAR ANTIBODIES CHARACTERIZATION OF PATTERNS OF ANTINUCLEAR ANTIBODIES B BY Y I IN ND DI IR RE EC CT T I IM MM MU UN NO OF FL LU UO OR RE ES SC CE EN NC CE E I IN N A AU UT TO OI IM MM MU UN NE E D DI IS SE EA AS SE ES S” ” is a bonafide work done by Dr.VIDHYAVATHI.V in partial fulfillment of the is a bonafide work done by Dr.VIDHYAVATHI.V in partial fulfillment of the r re eg gu ulla attiio on ns s o of f tth he e R Ra ajjiiv v G Ga an nd dh hii U Un niiv ve er rs siitty y o of f H He ea alltth h S Sc ciie en nc ce es s f fo or r tth he e a aw wa ar rd d o of f M M.. D D.. D De eg gr re ee e iin n P Pa atth ho ollo og gy y.. S Siig gn na attu ur re e o of f tth he e G Gu uiid de e D Da at te e : : D Dr r.. G GE EE ET TH HA AM MA AN NI I V V.. M MD D Professor and Head Professor and Head Place: Department of Pathology Place: Department of Pathology Kempegowda Institute of Medical Sciences Kempegowda Institute of Medical Sciences B Ba an ng ga allo or re e iii E EN ND DO OR RS SE EM ME EN NT T B BY Y T TH HE E H HE EA AD D O OF F T TH HE E D DE EP PA AR RT TM ME EN NT T//P PR RI IN NC CI IP PA AL L//H HE EA AD D O OF F T TH HE E I IN NS ST TI IT TU UT TI IO ON N T Th hiis s iis s tto o c ce er rttiif fy y tth ha att tth hiis s d diis ss se er rtta attiio on n ttiittlle ed d “ “D DE ET TE EC CT TI IO ON N A AN ND D C CH HA AR RA AC CT TE ER RI IZ ZA AT TI IO ON N O OF F P PA AT TT TE ER RN NS S O OF F A AN NT TI IN NU UC CL LE EA AR R A AN NT TI IB BO OD DI IE ES S B BY Y I IN ND DI IR RE EC CT T I IM MM MU UN NO OF FL LU UO OR RE ES SC CE EN NC CE E I IN N A AU UT TO OI IM MM MU UN NE E D DI IS SE EA AS SE ES S” ” is a bonafide work done by Dr. VIDHYAVATHI.V, under overall guidance of Dr. is a bonafide work done by Dr. VIDHYAVATHI.V, under overall guidance of Dr. GEETHAMANI V. M. D., Professor and Head, Department of Pathology, GEETHAMANI V. M. D., Professor and Head, Department of Pathology, K Ke em mp pe eg go ow wd da a I In ns sttiittu utte e o of f M Me ed diic ca all S Sc ciie en nc ce es s,, iin n p pa ar rttiia all f fu ullf fiillllm me en ntt o of f tth he e r re eg gu ulla attiio on ns s o of f the Rajiv Gandhi University of Health Sciences for the award of M. D. Degree in the Rajiv Gandhi University of Health Sciences for the award of M. D. Degree in P Pa atth ho ollo og gy y.. Seal and Signature of the HOD Seal and Signature of the Principal Seal and Signature of the HOD Seal and Signature of the Principal Dr. GEETHAMANI V. M. D. Dr. M. K. SUDARSHAN MD (BHU), FAMS Dr. GEETHAMANI V. M. D. Dr. M. K. SUDARSHAN MD (BHU), FAMS Professor and Head Principal Professor and Head Principal Department of Pathology Kempegowda Institute of Medical Sciences Department of Pathology Kempegowda Institute of Medical Sciences K Ke em mp pe eg go ow wd da a I In ns sttiittu utte e o of f M Me ed diic ca all S Sc ciie en nc ce es s B Ba an ng ga allo or re e.. B Ba an ng ga allo or re e.. D Da at te e: : D Da at te e: : Place: Place: Place: Place: iv COPYRIGHT COPYRIGHT DECLARATION BY THE CANDIDATE DECLARATION BY THE CANDIDATE I hereby declare that the Rajiv Gandhi University of Health Sciences, I hereby declare that the Rajiv Gandhi University of Health Sciences, K Ka ar rn na atta ak ka a s sh ha allll h ha av ve e tth he e r riig gh htts s tto o p pr re es se er rv ve e,, u us se e a an nd d d diis ss se em miin na atte e tth hiis s d diis ss se er rtta attiio on n // thesis in print or electronic format for academic / research purpose. thesis in print or electronic format for academic / research purpose. Date: Signature of the Candidate Date: Signature of the Candidate P Plla ac ce e: : N Na am me e:: D Dr r.. V VI ID DH HY YA AV VA AT TH H..V V  R Ra aj jiiv v G Ga an nd dh hii U Un niiv ve er rs siit ty y o of f H He ea allt th h S Sc ciie en nc ce es s,, K Ka ar rn na at ta ak ka a v ACKNOWLEDGEMENT Thanks giving is a pleasant job, but is nonetheless difficult when one sincerely tries to put it in words. These humble words of expression and gratitude cannot wholly convey my feelings. I express my deep sense of gratitude to Dr.GEETHAMANI.V, M.D., Professor and Head of the department, Department of Pathology, KIMS, Bangalore, for her valuable advice and constant guidance in the preparation of this work. I wish to profoundly thank my respected Professors of Pathology, Dr.B.V.Suguna , M.D., Dr.V.Kusuma , M.D.,D.C.P., Dr.R.Rangaswamy, M.D., Dr.S.R.Niveditha M.D., D.N.B, for their help and kindly suggestion. I sincerely thank Associate professors Dr.Savithri Ravindra, D.C.P., D.N.B, Dr.Suja AjoyKumar M.D.,D.N.B, for their encouragement and support. I sincerely thank Dr. Thejasvi Krishnamurthy M.D., Dr. Sruthi Prasad M.D, Dr. Manjula C.P M.D., Dr. Chethana Mannem M.D., Dr. Preetha D Prabhu D.C.P, Dr. Gayathri D.C.P, Dr. Anagha Joshi, D.C.P, Dr. Venkatesh Prasad M.B.B.S; for their encouragement and support. I extend my heartful gratitude to Dr. M.K.Sudarshan, M.D., Dean & Principal, KIMS, Bangalore, Bangalore for his support. I am grateful to Dr. Jayaram N., MD, Anand Diagnostics Laboratory and Dr. Ravikumar H.N., MD, RV laboratory for permitting me to learn the Immuno- fluorescence technique in their esteemed laboratories. vi I am thankful to all the non-teaching staff, department of Pathology, KIMS, Mrs.Padma, Mr.Bomme Gowda, Mr.Krishnappa, Mr.Lakshman Gowda, Mrs. Sunandamma and hematology technicians for their skilled technical assistance. Completion of the study would not have been possible without their constant assistance. It is beyond words to express thanks to my husband, my parents and all my colleagues for their unfailing support. I am grateful to all the patients who despite their suffering co-operated in completing the project. D Da atte e:: S Siig gn na attu ur re e o of f tth he e C Ca an nd diid da atte e P Plla ac ce e:: N Na am me e:: D Dr r.. V VI ID DH HY YA AV VA AT TH HI I..V V.. vii L LI IS ST T O OF F A AB BB BR RE EV VI IA AT TI IO ON NS S U US SE ED D ACA Anticentromere antibody AI disease Autoimmune diseases ANA Antinuclear antibody anti-RNP Anti –ribonucleoproteins anti-Sm Anti- Smith anti-topo I Anti-DNA topoisomerase I antibody anti- Anti-RNA polymerases I,II and III RNAP APL Anti -phospholipid antibodies ARD Autoimmune Rheumatic diseases B Bone marrow derived lymphocyte CAR American college of Rheumatology CCP Cyclic citrullinated peptide CDC The centers for disease control & prevention CENP-F Centromere protein CLIF Crithidia lucilia immunofluorescence CLSI Clinical & laboratory institute CREST Calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia CTD Connective tissue disease DNA Deoxy ribonucleic acid ds DNA Double-stranded DNA DSSc Diffuse cutaneous Systemic sclerosis H Histidine HLA Human leukocyte antigen EIT Electroimmuno transference ELISA Enzyme linked immune sorbent assay viii FANA Fluorescent antinuclear antibody test 2 GPI 2 glycoprotein I HEp -2 Human epithelial cells Ig Immunoglobulins IL1 Interleukin 1 ILAR International League Of Associations For Rheumatology IIF Indirect immunofluorescence JIA Juvenile idiopathic arthritis JRA Juvenile Rheumatoid arthritis LSSc Limited cutaneous Systemic sclerosis MCTD Mixed connective tissue disease MHC Major histocompatibilty complex nDNA Native DNA NuMA Nuclear mitotic apparatus PBS Phosphate buffer saline PCNA Proliferating cell nuclear antigen RA Rheumatoid arthritis RIA Radioimmunoassay RNA Ribonucleic acid SLE Systemic lupus erythematosus Sn RNP Small nuclear ribonuclear protein SSc Systemic sclerosis Ss DNA Single stranded DNA Sub ac.LE Sub acute lupus erythematosus T cells Thymus derived lymphocye T T helper cells H TLR Toll like receptors U Uridine UV Ultra violet ix ABSTRACT BACKGROUND AND OBJECTIVES This study was intended to detect ANA in serum samples suspected to have Autoimmune(AI) diseases and to characterise specific patterns of staining of ANA with positive screening test .The usefulness of Indirect Immunofluorescence(IIF) technique as a screening procedure and frequency of occurrence of specific IIF pattern in various connective tissue disorders were also assessed. METHODS Antinuclear antibody detection by IIF method on Euroimmun ANA kits containing monkey liver tissue and human epithelial-2 (HEp-2) cells was conducted in a total of 300 consecutive sera of patients with suspected autoimmune diseases referred to KIMS Hospital and Research Center, Bangalore .The test was done over a period of 18 months from January 2010 to July 2011. Those patients with Systemic lupus erythematosus were further studied by Crithidia lucilia immunofluorescence (CLIF) assay for double stranded(ds) DNA for confirmation. RESULTS Out of 300 cases of suspected AI diseases, 35(12%) cases were diagnosed as Autoimmune disease.ANA positivity was observed in 23 (66%) cases of AI diseases. Nucleus coarse granular pattern was the most common pattern in these cases. Double stranded(ds) DNA test by CLIF method was done for suspected SLE cases and 11(83%) cases turned to be positive for ds DNA. x INTERPRETATION AND CONCLUSION We found that ANA test by IIF method serves as a simple, cheap and reliable screening test for Autoimmune diseases. The IIF staining patterns gives a clue to the diagnosis of individual disease. It can also be used to know the disease activity in certain AI diseases like SLE and Sjogren’s syndrome. KEYWORDS Antinclear antibody, Indirect immunofluorescence ,Autoimmune diseases, Nucleus coarse granular pattern, ds DNA, CLIF. xi T TA AB BL LE E O OF F C CO ON NT TE EN NT TS S P Pa ag ge e N No os s.. 1. INTRODUCTION 1 1. INTRODUCTION 1 2 2.. A AI IM MS S A AN ND D O OB BJ JE EC CT TI IV VE ES S 4 4 3. REVIEW OF LITERATURE 5 3. REVIEW OF LITERATURE 5 4 4.. M ME ET TH HO OD DO OL LO OG GY Y 3 33 3 5 5.. O OB BS SE ER RV VA AT TI IO ON N A AN ND D A AN NA AL LY YS SI IS S 3 38 8 6. DISCUSSION 76 6. DISCUSSION 76 7 7.. C CO ON NC CL LU US SI IO ON N 8 84 4 8. SUMMARY 86 8. SUMMARY 86 9 9.. B BI IB BL LI IO OG GR RA AP PH HY Y 8 88 8 1 10 0.. A AN NN NE EX XU UR RE ES S 95  PROFORMA 95  PROFORMA 97  MASTER CHART 97  MASTER CHART 1 11 12 2  K KE EY Y T TO O T TH HE E M MA AS ST TE ER R C CH HA AR RT T xii L LI IS ST T O OF F T TA AB BL LE ES S P Pa ag ge e N No os s.. 1. 38 1. Total number of Autoimmune(AI) diseases 38 2 2.. 4 40 0 Percentage distribution of AI diseases 3. 42 3. Age distribution in AI diseases 42 4 4.. 4 44 4 Age distribution in individual AI diseases 5 5.. 4 47 7 Gender distribution in AI diseases 6. 49 6. Gender distribution in individual type of AI diseases 49 7 7.. 5 51 1 ANA positive Autoimmune diseases 8. 53 8. Total no. Of ANA positive cases in each AI disease 53 9. 56 9. Various nuclear fluorescence patterns observed in our study 56 1 10 0.. 5 58 8 Characterisation of patterns in various AI disorders. 11. 61 11. Double stranded(ds) DNA in SLE cases. 61 1 12 2.. 6 63 3 Total no. of AI disorders with skin involvement. 13. 65 13. Characterisation of patterns of nuclear fluorescence in AI diseases 65 with skin involvement. xiii L LI IS ST T O OF F F FI IG GU UR RE ES S Page Page Nos. Nos. 1 1.. Olympus Fluorescent Microscope (Model-BX41) 6 68 8 2 2.. Euroimmun ANA Kits and other requirements 6 68 8 3. A SLE case showing characteristic “Butterfly rash” on malar area. 69 3. 69 4 4.. A SLE case showing Psoriasiform eruptions on the back of forearm. 6 69 9 5 5.. A SLE case showing vasculitic ulcers on the lowerlimbs 7 70 0 6. A Scleroderma case showing taut, shiny skin and mask-like face. 70 6. 70 7. A Scleroderma case showing taut skin in the face and neck. 71 7. 71 8 8.. Nucleus Homogenous,HEp-2 Cells, Serum Dilution 1:100, X400 7 72 2 9. Nucleus Homogenous, Monkey Liver, Serum Dilution 1:100, X 400 72 9. 72 10. Nucleus Coarse Granular,HEp-2 Cells, Serum Dilution 1:100, X400 73 10. 73 1 11 1.. Nucleus Coarse Granular Pattern In Monkey Liver, Serum Dilution 73 73 1:100, X 400 12. Nucleus Fine Granular ,HEp-2 Cells, Serum Dilution 1:100, X 400 74 12. 74 1 13 3.. Nucleoli Homogenous,HEp-2 Cells,Serum 1:100,X 400 7 74 4 1 14 4.. Nuclear Rim, HEp-2 Cells; Serum Dilution 1:100, X 400 7 75 5 15. ds DNA, Crithidia Luciliae,Serum Dilution 1:50,X400 75 15. 75 xiv L LI IS ST T O OF F G GR RA AP PH HS S P Pa ag ge e Nos. Nos. 1 1.. Total number of Autoimmune(AI) diseases 3 39 9 2. 2. Percentage distribution of AI diseases 41 41 3. 3. Age distribution in AI diseases 4 43 3 4 4.. Age distribution in individual AI diseases 4 45 5 5. 5. Gender distribution in AI diseases 48 48 6 6.. Gender distribution in individual type of AI diseases 5 50 0 7. 7. ANA positive Autoimmune diseases 52 52 8. 8. Total no. Of ANA positive cases in each AI disease 54 54 9 9.. Various nuclear fluorescence patterns observed in our study 5 57 7 10. 10. Characterisation of patterns in various AI disorders. 59 59 1 11 1.. Double stranded(ds) DNA in SLE cases. 6 62 2 12. 12. Total no. of AI disorders with skin involvement. 64 64 1 13 3.. Characterisation of patterns of nuclear fluorescence in AI diseases 6 66 6 with skin involvement. xv INTRODUCTION Autoimmune (AI) diseases form a heterogeneous group of illnesses characterized by humoral or cell-mediated immune reactions against one or more of the body’s own constituents. Clinicians classify autoimmune diseases as systemic or 1 organ specific .Systemic autoimmune diseases display autoantibodies directed against nuclear or cytoplasmic molecules that participate in DNA replication, DNA transcription and the translation of messenger RNA. Organ-specific autoimmune 1 diseases exhibit autoantibodies directed against an organ or related organs . It has been suggested that autoimmune diseases comprise different phases 2 (phase 1 to 4) . In the initial phase, which can occur many years before the diagnosis, patients are asymptomatic and have no serum autoantibodies , but they are carriers of a gene set (i.e., HLA, IgA deficiency, Component deficiencies) that predisposes them to a given autoimmune disease. Specific autoantibodies appear in the serum in the second phase; at this stage, the patient is still asymptomatic or pauci symptomatic. Finally, in the third phase, the disease is clinically manifested with signs and symptoms that generally bring the patient to the attention of the clinician and allow a 2 definite diagnosis to be determined . AI diseases are characterised by the presence of antinuclear antibodies (ANA). ANAs are a heterogenous group of circulating Immunoglobulins (Igs) such as IgM, IgG & IgA. These Igs react with whole nucleus or nuclear components such as nuclear 3 proteins, DNA or histones in host tissues . ANAs are used to screen the AI diseases in symptomatic or paucisymptomatic patients. In autoimmune rheumatic diseases (ARDs), the autoantibodies provide classification and diagnostic criteria for these diseases. ANAs are used both to define the prognosis and as markers for disease 3 activity . 1 Generally, ANAs have no organ or species specificity and are capable of cross reacting with nuclear material from humans (eg. human leukocytes) or various animal tissues (eg.rat liver, mouse, kidney). Demonstration of ANAs in laboratory testing can indicate various systemic AI connective tissue disorders (eg.SLE, Multiple connective tissue disorder (MCTD), Systemic sclerosis (SSc), Sjogren syndrome, Rheumatoid arthritis (RA) and Polymyositis). These disease states are characterised by antibodies that react with different nuclear components such as double stranded (ds) DNA, single 3 stranded (ss) DNA and Smith antigen . ANAs are directed against several nuclear antigens and can be grouped into 3 four categories : 1) Antibodies to DNA, 2) Antibodies to histones, 3) Antibodies to nonhistone proteins bound to RNA and 4) Antibodies to nucleolar antigens. In recent years, numerous methods like radioimmunoassay (RIA), ELISA, Electroimmunotransference (EIT) or Western blot have been developed for the 4 identification of ANA in the serum . The most common screening test is Indirect 5 Immunofluorescence method (IIF) on rodent liver or human epithelial (HEp2) tissue , although ELISA tests are available.It is a simple, cheap, screening procedure which can identify antibodies that bind to a variety of nuclear antigens, including DNA, 6 RNA, and proteins (collectively called generic ANAs) . There are only three diseases that include or require a positive antinuclear antibody test in their diagnostic criteria. The first disease is SLE. It is generally accepted that greater than 95 % of patients with this disease will have a positive ANA test. In addition, virtually 100% of patients with MCTD and drug-induced lupus 6 erythematosus have a positive ANA test . 2 According to the III National Consensus Standards for ANA Records, more than 25 possible patterns of fluorescence are described. Each one of them may reflect 7 a given antigen expression recognized by its autoantibody. Fluorescence patterns Different fluorescent staining patterns are reported which give clues as to the 8 significance of the ANA and type of Connective tissue disease (CTD) . 1. Nuclear patterns: Homogeneous, speckled (fine and coarse), 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 are of clinical significance. ANA test is important in conjunction with clinical evaluation and in the absence of symptoms and signs of a CTD; a positive ANA test only confounds the diagnosis. A positive ANA test may rarely be seen in healthy individuals, particularly the elderly or in a wide range of 8 diseases other than CTD, where it has no diagnostic or prognostic value . 3 AIMS AND OBJECTIVES 1. To detect Anti nuclear antibody (ANA) in serum samples suspected to have Autoimmune diseases. 2. To detect specific pattern of staining of ANA with positive IIF screening test. 3. To assess the usefulness of Immunofluorescence technique as a screening procedure for various connective tissue disorders. 4 REVIEW OF LITERATURE AUTOIMMUNE DISEASES Autoimmune (AI) disease processes are due to immune responses generated by the body against its own cells or tissues. These are among the 10 leading causes of 9 death among women in all age groups up to 65 . At least 2% of the general population is affected. In early 1900, Ehrlich described the breakdown of self-tolerance associated with autoimmune diseases as “horror autotoxicus” which means fear of 10 self-poisoning . Factors which predispose to the development of autoimmunity include genetics factors, age of the individual and environmental factors. Epidemiologic studies have demonstrated that genetic factors are crucial determinants 11 of susceptibility to autoimmune disease. Various theories to explain the mechanism of autoimmune diseases: 1. Forbidden clone theory 2. Clonal anergy theory 3. Sequestered antigen theory and 4. Immunologic deficiency theory 1. Forbidden clone theory: The antibodies on the surface of immune cells serve as receptors for specific antigens, which cause the corresponding cells to be stimulated to eliminate the stimulating antigens. This leads to proliferation of the stimulated cells with the establishment of clones of identical cells, some of which secrete antibodies while others become memory cells. If self antigens are not recognized during fetal life and 5