R



|RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

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|1. |Name of Candidate and Address |Dr.VIDHYAVATHI |

| | |15/193,Ex-service men colony, Golden Rock, Tiruchy-620004, TN. |

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| |Name of the Institution |KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES, BANGALORE – 560 070 |

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|3. |Course of the Study & Subject |M.D PATHOLOGY |

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|4. |Date of Admission to Course |28.04.09 |

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| | |DETECTION AND CHARACTERIZATION OF PATTERNS OF |

| | |ANTINUCLEAR ANTIBODIES BY INDIRECT IMMUNOFLOURESCENCE IN AUTOIMMUNE DISEASES. |

|5. |Title of the Topic | |

|6. |Brief Resume of the Intended Work |

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| |6.1 Need for the Study: |

| |Immunofluorescence techniques(IF) have been used for over 65 years to localize antigenically distinct molecules in tissue sections for |

| |microscopic study.The indirect immunofluorescence was introduced by Weller and Coons in 1954 .They originally studied the interaction |

| |between antigen (herpes zoster virus monolayer) and patients’ serum containing antibody to this virus.1 In1957 ,George Friou introduced |

| |immunofluorescence –anti nuclear antibody (IF-ANA). Since then, this method is widely used for the diagnosis of connective tissue |

| |diseases. It is cheap and easy to perform with high sensitivity. |

| |Keeping this in mind, we have decided to start the procedure as a routine screening procedure for the diagnosis of certain common |

| |autoimmune diseases(AI) like Rheumatoid arthritis(RA), SLE, Sjogrens disease, Systemic sclerosis, Drug induced lupus(DLE), Hashimotos |

| |disease, Graves disease. Different autoimmune diseases have various patterns of immunofluorescent staining. ANA is present in low titers |

| |in approximately 5-15% of normal individuals and it also increases with age. In such a situation, clinical correlation is very important.|

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| |While reporting ,three parameters are important: |

| |Pattern of fluorescence |

| |Substrate used |

| |The titer of ANA of a positive test. |

| |Our study includes the patients with signs and symptoms of AI diseases and to screen them for the presence of ANA. Our study also |

| |includes the study of various patterns of fluorescence of ANA in different autoimmune diseases .If required, to be more specific about |

| |ANA, ANA profile test will be done. Also anti ds DNA antibody study using Crithidia luciliae substrate will be done if needed by the |

| |clinician. |

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| |6.2 Review of literature: |

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| |Immunofluorescence technique uses specific antibody to detect antigenic differences at molecular level. The combination of antibody with |

| |the specific antigen does not lead to a visible change and therefore a readily identifiable label must be irreversibly bound to the |

| |antibody so that its localization can be recognized. In immunofluorescence technique, this label takes the form of a flourochrome, |

| |eg;flourescein or rhodamine which absorb radiation and emit the radiation of a different wavelength within the visible spectrum. 1 |

| |Immunofluorescence technique has become a valuable diagnostic tool in the study of AI diseases as it is extremely sensitive. The various |

| |patterns observed in IF staining are: |

| |Homogenous pattern |

| |Peripheral pattern |

| |Speckled pattern |

| |Nucleolar pattern |

| |Homogeneous or diffuse nuclear staining usually reflects antibodies to chromatin, histones and, occasionally, double-stranded DNA. |

| |Rim or peripheral staining patterns are most commonly indicative of antibodies to double-stranded DNA. |

| |Speckled pattern refers to the presence of uniform or variable-sized speckles. This is one of the most commonly observed patterns of |

| |fluorescence and therefore the least specific.It reflects the presence of antibodies to non-DNA nuclear constituents. |

| |Examples include Sm antigen, ribonucleoprotein, and SS-A and SS-B reactive antigens . |

| |Nucleolar pattern refers to the presence of a few discrete spots of fluorescence within the nucleus and represents antibodies to |

| |nucleolar RNA. This pattern is reported most often in patients with systemic sclerosis.2 |

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| |Indirect immunoflourescence microscopy(IIFM) has been a popular and sensitive |

| |method for detecting anti-TPO(AMA).Three patterns of immunoflourescence are described when anti-Tg is present:1.floccular pattern |

| |2.Dull colloid spaces but bright peripheral flourescence |

| |3.Diffuse,bright,uniformly staining colloid in a “ground glass ”pattern.3 |

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| |Antinuclear Antibodies in Various Autoimmune Diseases: |

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| |DISEASES |

| |ANTIGENS |

| |ANTIBODY-ANA2 |

| |PATTERN OF FLUORESCENCE4 |

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| |SLE & DLE |

| |1.Native DNA |

| |2.Histone |

| |3.Smith Ag |

| |4.Ribonuclear protein |

| |(RNP) |

| |1.Anti ds DNA |

| |2.Anti Histone |

| |3.Anti Smith |

| |4.Nuclear RNP |

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| |1 .Homogenous(MC) |

| |2.Peripheral(active |

| |stage of ds) |

| |3.speckled |

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| |RHEUMATOID ARTHRITIS |

| |Many nuclear antigens |

| |(DNA, RNA,proteins) |

| |Generic ANA |

| |1.Homogenous |

| |2.speckled |

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| |SJOGREN’S DISEASE |

| |1.RNP |

| |2.RNP |

| |SS-A(Ro) |

| |SS-B(La) |

| |1.Speckled(MC) |

| |2.Homogenous |

| |3.Peripheral |

| |4.nucleolar |

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| |SYSTEMIC SCLEROSIS |

| |1.DIFFUSE |

| |2.LIMITED |

| |1.Many nuclear antigens |

| |2.DNA-topoisomerase I |

| |3.Centromeric proteins |

| |1.Generic ANA |

| |2.SCL-70(diffuse) |

| |3.Anticentromere |

| |(limited) |

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| |Nucleolar(MC) |

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| |HASHIMOTO’S DISEASE |

| |Microsomal fraction of |

| |thyroid |

| |epithelial cell |

| |Anti TPO |

| |Granular pattern in the cytoplasm |

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| |GRAVES DISEASE |

| |Thyroglobulin(Tg) |

| |Anti Tg |

| |1.Floccular pattern |

| |2.Dull colloid spaces |

| |but bright peripheral |

| |fluorescence |

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| |Laurino C C, Lora P S,Brenol J C T,Xavier R M-they evaluated the presence of patterns of ANA by Indirect Immunoflourescence tecnique in a|

| |uiversity hospital and found nuclear fine speckled pattern as the most frequent pattern.5 |

| |Hayashi N et al , compared new Enyme immuno assay and Immunoflourescence assay in 307 patients with connective tissue disease and 492 |

| |healthy individuals.For 258 out patient department patients at serum dilution 1/40 ,sensitivity and specificity |

| |of IF method |

| |are 92% and 65% respectively |

| |At serial dilutions 1/160 , sensitivity and specificity of IF method are 81% and 87% respectively. Thus Immunofluorescent assay with |

| |higher dilutions show an increased specificity.6 |

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| |6.3 Objectives of the Study: |

| |To detect ANA in serum samples suspected to have autoimmune diseases7. |

|7. |To detect specific pattern of staining of ANA with positive screening test. |

| |To assess the usefulness of IF technique as a screening procedure for various connective tissue disorders like RA, SLE, Sjogrens disease,|

| |Systemic sclerosis, Drug induced lupus(DLE), Hashimotos disease, Graves disease. |

| |Materials and methods: |

| |7.1 Source of Data: |

| |Suspected cases of AI diseases referred to the departments of Pathology by the department of Medicine,Orthopaedics and Dermatology, |

| |during the period of JANUARY 2010 to JUNE 2011. |

| |7.2 Method of Collection of data: |

| |The procedure requires 2ml of patients’ serum, phosphate buffer saline(PBS), substrate, fluorescence labeled conjugate, Fluorescent |

| |microscope. The substrate most frequently used is HEp2 cells (Human epithelial cells).Other common substrates used are mouse kidney and |

| |stomach and Crithidia luciliae. The procedure for detecting anti ds DNA and other ANA differs only in the substrates used. Crithidia |

| |luciliae,a haemoflagellate is used as a substrate for detecting Anti ds DNA and HEp2cells are used in detection of other ANA. |

| |The serum diluted 1/40 in phosphate buffer saline(PBS) was overlaid onto fixed HEp2 cells for 30 minutes at room temperature. Slides |

| |were washed twice for 5 minutes each with PBS overlaid with Fluorescence labeled conjugate which is antihuman IgG and incubated for 30 |

| |minutes. After washing twice, the coverslip was placed over the slide and the slides were read using Fluorescent microscope at x40 power.|

| |The fluorescence intensity was scored semi quantitatively from 1+ to 5+ relative to intensity of a negative and positive control 4+. |

| |7.3 Sampling: |

| |Purposive sampling. |

| |Study design: |

| |Descriptive study. |

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| |7.4 Inclusion Criteria: |

| |Patients referred to the department of Pathology for ANA screening. |

| |Patients referred to the department of Pathology for FNAC of diffuse goiter who are reported as Hashimotos disease or Graves disease by |

| |our department. |

| |7.5 Exclusion Criteria: |

| |Patients with sero negative arthritis. |

| |Cases with positive staining pattern but have no clinical signs and symptoms of connective tissue diseases. |

| |AI diseases where ANA antibody is not produced and is not used as a diagnostic screening procedure. |

| |7.6 Place of Study: |

| |Department of Pathology- Kempegowda institute of medical sciences, Hospital and Research Center, Bangalore. |

| |7.7 Study Period: |

| |January 2010 to July 2011. |

| |7.8 Statistical Methods Involved: |

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| |Data collected will be analyzed statistically using frequency distribution and will be |

| |presented in the form of graphs and charts. |

| |7.9 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please |

| |describe briefly. |

| |It does not require any investigations. |

| |No interventions in the animals. |

| |7.10 Has ethical clearance been obtained from your institution in case of 7.9? |

| |Yes. |

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| |List of references: |

| |Hladik C.L ,White C.L , Immunofluorescent Techniques. In: Theory & Practice Of Histological Techniques, 6thedition, Bancroft J.D, Gamble|

| |M, China, Churchill Livingstone.2008:517-26. |

| |Abbas A. K, Diseases Of Immunity. In :Robbins And Cotran, Pathologic Basis Of Disease,7th Edition,Kumar,Abbas and Fausto. New Delhi, |

| |Saunders. 2004:193-268. |

| |Bylund D J, Nakamura R M ,Organ Specific Autoimmune Diseases. In: Clinical Diagnosis And Management By Laboratory Methods, Henry J B, |

| |20th Edition, New Delhi,Harcourt.2003:1000-15. |

| |Turgeon M.L , Systemic Lupus Erythematosus. In: Immunology And Serology In Laboratory Medicine, Turgeon M.L, 3rd Edition, Philadelphia, |

| |Mosby. 2003:401-420. |

| |Laurino C C F C , Lora P S, Brenol J C T, Grutcki D M, Xavier R M. Experience Of An University Hospital On The Implementation Of 1&2 |

| |Brazilian Consensus For Standardization Of ANA In Hep-2 Cells. J Rev Bras Rheumatol 2009;49(2):110-20. |

| |Hayashi N et al. Detection Of ANA By Use Of An Enzyme Immune Assay With Nuclear HEp-2 Cell Extract And Recombinant Antigens: Comparison |

| |With Immunofluorescence Assay In 307 Patients. J Clinical Chemistry 2001; 47(9) :1649-1659. |

| |Peene I, Meheus L, Veys E M, Keyser FD. Detection And Identification Of Anti Nuclear Antibodies(ANA) In A Large And Consecutive Cohort Of|

| |Serum Samples Referred For ANA Testing. J Ann Rheum Dis 2001;60:1131-1136. |

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| |9.1 Signature of the Candidate: |

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| |9.2Remarks of the Guide: |

| |Indirect immunofluorescent technique is a widely used method. It is known for its high sensitivity. It is easy to perform , time saving |

| |procedure and cheap compared to the other advanced techniques.It can be effectively done as a routine screening test to diagnose and to |

| |monitor the prognosis of various auto immune diseases including Hashimotos disease. |

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| |10.1 Name&Designation of Guide: DR.GEETHAMANI.V M.D., |

| |Professor and Head of the department, |

| |Department of Pathology |

| |KIMS, Bangalore |

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| |10.2Signature: |

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| |10.3 Head of the Department Dr. GEETHAMANI V M.D |

| |Professor & Head Of The Department, |

| |Department of Pathology, |

| |KIMS, Bangalore. |

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| |10.4 Signature: |

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| |10.5 Remarks of Chairman & Principal: |

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|10. | |

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