R
|RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA
BANGALORE
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
| |
| | | |
| | | |
| | | |
|1. |Name of Candidate and Address |Dr.VIDHYAVATHI |
| | |15/193,Ex-service men colony, Golden Rock, Tiruchy-620004, TN. |
| | | |
| |Name of the Institution |KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES, BANGALORE – 560 070 |
|2. | | |
| | | |
|3. |Course of the Study & Subject |M.D PATHOLOGY |
| | | |
|4. |Date of Admission to Course |28.04.09 |
| | | |
| | |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 |
| | |
| |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.|
| | |
| | |
| | |
| |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. |
| | |
| |6.2 Review of literature: |
| | |
| |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 |
| | |
| | |
| | |
| | |
| |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 |
| | |
| |Antinuclear Antibodies in Various Autoimmune Diseases: |
| | |
| |DISEASES |
| |ANTIGENS |
| |ANTIBODY-ANA2 |
| |PATTERN OF FLUORESCENCE4 |
| | |
| |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 |
| | |
| |1 .Homogenous(MC) |
| |2.Peripheral(active |
| |stage of ds) |
| |3.speckled |
| | |
| |RHEUMATOID ARTHRITIS |
| |Many nuclear antigens |
| |(DNA, RNA,proteins) |
| |Generic ANA |
| |1.Homogenous |
| |2.speckled |
| | |
| |SJOGREN’S DISEASE |
| |1.RNP |
| |2.RNP |
| |SS-A(Ro) |
| |SS-B(La) |
| |1.Speckled(MC) |
| |2.Homogenous |
| |3.Peripheral |
| |4.nucleolar |
| | |
| |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) |
| | |
| | |
| |Nucleolar(MC) |
| | |
| |HASHIMOTO’S DISEASE |
| |Microsomal fraction of |
| |thyroid |
| |epithelial cell |
| |Anti TPO |
| |Granular pattern in the cytoplasm |
| | |
| |GRAVES DISEASE |
| |Thyroglobulin(Tg) |
| |Anti Tg |
| |1.Floccular pattern |
| |2.Dull colloid spaces |
| |but bright peripheral |
| |fluorescence |
| | |
| | |
| |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 |
| | |
| | |
| |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. |
| | |
| |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: |
| | |
| |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. |
| | |
| | |
| | |
| |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. |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| |9.1 Signature of the Candidate: |
| | |
| | |
| | |
| |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. |
|8. | |
| | |
| | |
| | |
| | |
| |10.1 Name&Designation of Guide: DR.GEETHAMANI.V M.D., |
| |Professor and Head of the department, |
| |Department of Pathology |
| |KIMS, Bangalore |
| | |
| | |
| | |
| |10.2Signature: |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| |10.3 Head of the Department Dr. GEETHAMANI V M.D |
| |Professor & Head Of The Department, |
| |Department of Pathology, |
| |KIMS, Bangalore. |
| | |
| | |
| | |
| |10.4 Signature: |
| | |
| | |
| | |
| | |
| |10.5 Remarks of Chairman & Principal: |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
|9. | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
|10. | |
| | |
| | |
................
................
In order to avoid copyright disputes, this page is only a partial summary.
To fulfill the demand for quickly locating and searching documents.
It is intelligent file search solution for home and business.
Related searches
- dod 7000.14 r vol 12 ch 7
- dod fmr 7000.14 r vol 8
- dod 7000.14 r volume 2a
- dod 7000.14 r volume 7a 2018
- dod 7000.14 r 2019
- dod 7000.14 r volume 9
- dod 7000.14 r volume 6
- dodfmr 7000.14 r vol 5
- dod fmr 7000.14 r volume 5
- statistically significant correlation r value
- dod fmr 7000.14 r volume 8
- dod 7000.14 r volume 2a chapter 1