<p> RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE .</p><p>ANNEXURE II</p><p>PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION</p><p>1. NAME OF THE CANDIDATE AND Dr. SONAL GROVER POST GRADUATE STUDENT, ADDRESS DEPARTMENT OF ORAL PATHOLOGY AND (IN BLOCK LETTERS) MICROBIOLOGY, BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE-577004, KARNATAKA.</p><p>2. NAME OF THE INSTITUTION BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE-577004, KARNATAKA.</p><p>3. COURSE OF STUDY MASTER OF DENTAL SURGERY ORAL PATHOLOGY AND MICROBIOLOGY. AND SUBJECT</p><p>4. DATE OF ADMISSION TO THE 21/04/2009 COURSE</p><p>5. TITLE OF THE TOPIC: “ COMPARATIVE STUDY FOR SELECTIVITY OF MICRONUCLEI IN ORAL EXFOLIATED CELLS OF POTENTIALLY MALIGNANT LESIONS USING FEULGEN STAIN, PAPANICOLAOU STAIN AND HAEMOTOXYLIN & EOSIN STAIN: A CLINICOCYTOPATHOLOGICAL STUDY.” 6 BRIEF RESUME OF INTENDED WORK:</p><p>. 6.1 Need for the study</p><p>“Prevention is better than cure"</p><p>Many oral cancers are preceded by clinically evident premalignant mucosal changes that</p><p> give a warning of risk and present an opportunity for detection and preventive measures. The key</p><p> to diagnosis is the early detection of mucosal changes that may represent disease and not</p><p> variations of normal. Cytological study of oral cells is a nonaggressive technique that is well</p><p> accepted by the patient, and is therefore an attractive option for the early diagnosis of potentially</p><p> malignant lesions.1</p><p>The analysis of micronuclei (MN) has gained increasing popularity as an in vitro</p><p> genotoxicity test and a biomarker assay for human genotoxic exposure and effect. In comparison</p><p> with chromosomal aberrations (CA), the scoring of MN is simpler, requires shorter training and is</p><p> less time consuming. In principle, the MN assay can be expected to be more sensitive than the CA</p><p> assay, because of the increased statistical power brought about by the fact that the number of cells</p><p> analysed can easily be increased to thousands when only a hundred or a few hundred cells are</p><p> usually scored for chromosomal aberrations.2</p><p>Micronucleus (MN) is defined as microscopically visible, round or oval cytoplasmic</p><p> chromatin mass next to the nucleus. Micronuclei originate from aberrant mitoses and consist of</p><p> eccentric chromosomes, chromatid fragments or whole chromosomes that have failed to be</p><p> incorporated into the daughter nuclei during mitosis.3 </p><p>Radiology and Oral Pathology and Microbiology, Bapuji Dental College and Hospital,</p><p>Davangere as outpatients with lesions clinically diagnosed to be potentially malignant.</p><p>7.2 Method of Collecting Data (Including Sampling Procedures, If Any):</p><p>Study sample will include oral cytological smear from patients with lesions clinically diagnosed to be potentially malignant. Healthy subjects with clinically normal oral mucosa will be taken as controls. The written consent to carry out the cytological smears which are required for the study will be obtained, after the necessary instructions. Ethical clearance has been obtained for the same.</p><p>INCLUSION CRITERIA:</p><p>Subjects included in the study will be those clinically diagnosed as one of the following potentially malignant lesions of the oral cavity:</p><p>Leukoplakia, Erythroplakia, Oral submucous fibrosis and Lichen planus.</p><p>EXCLUSION CRITERIA:</p><p>Patients with provisional or confirmed diagnosis of any cancer will not be included in the study.</p><p>Study sample will be divided into two groups:</p><p>Group 1: Control group comprise of 10 healthy subjects with clinically normal oral mucosa.</p><p>Group 2: Comprise of 40 patients with clinically diagnosed as potentially malignant lesions of the oral cavity. 8. Three cytosmears from lesions of the subjects will be obtained as follows:</p><p>The subjects will be asked to rinse their mouth with water and a cytobrush will be used to obtain</p><p> exfoliated cells from the oral mucosa. The samples will be transferred to three dry glass slides, to</p><p> ensure an adequate harvest of cells. Smears will be air dried and fixed with 95% ethanol spray.</p><p>Smears will be separately stained with following stains:</p><p>1. Feulgen stain</p><p>2. Papanicolaou stain</p><p>3. Haemotoxylin & Eosin stain.</p><p>For designating an extra nuclear body as micronucleus, the following criteria given by</p><p>Tolbert3 will be considered and 1000 cells will be scored to determine the micronuclei</p><p> frequency:</p><p>(a) Rounded smooth perimeter suggestive of a membrane. </p><p>(b) Less than a third the diameter of the associated nucleus, but large enough to discern shape</p><p> and color.</p><p>(c) Staining intensity similar to that of the nucleus. </p><p>(d) Texture similar to that of nucleus. </p><p>(e) Same focal plane as nucleus.</p><p>(f) Absence of overlap with, or bridge to, the nucleus.</p><p>Statistical analysis: Data obtained will be statistically analyzed by ANOVA for comparison between the staining procedures. Groupwise comparison will be made by Wilcoxon’s ranksum test (Man-</p><p>Whitney U test). </p><p>7.3: Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly. </p><p>Yes, oral cytosmears will be taken from patients with clinically diagnosed potentially malignant lesions for cases and cytosmears from subjects with clinically normal oral mucosa will be taken for controls.</p><p>7.4: Has ethical clearance been obtained from your institution in case of 7.3. </p><p>Yes</p><p>LIST OF REFERENCES:</p><p>1. Epstein BJ, Gorsky M, Fischer D, Gupta A, Epstein M, Elad S. A Survey of the Current Approaches to the Diagnosis and Management of Oral Premalignant lesions. J Am Dent Assoc 2007; 138:1555-1562.</p><p>2. Norppa H and Falck G. What do micronuclei contain? Mutagenesis 2003; 18(3):221-233. 3. Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmuller S. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: The HUMN project perspective on current status and knowledge gap. Mutat Res 2008; 659(1-2):93-108.</p><p>4. Nersesyan A, Kundi M, Atefie K, Hermann RS, Knasmuller S. Effect of Staining Procedures on the Results of Micronucleus Assays with Exfoliated Oral Mucosa Cells. Cancer Epidemiol Biomarkers Prev 2006; 15(10):1835-1840. </p><p>5. Casartelli G, Monteghirfo S, De Ferrari M, Bonatti S, Scala M, Toma S et al . Staining of</p><p> micronuclei in squamous epithelial cells of human oral mucosa. Anal Quant Cytol Histol 1997; 19(6):475-81. </p><p>6. Belein MAJ, Copper PM, Braakhus MJB, Snow BG, Baak APJ. Standardization of counting micronuclei: definition of a protocol to measure genotoxic damage in human exfoliated cells. Carcinogenesis 1995; 16(10):2395-2400. </p><p>7. Mollaoglu N, Cowpe GJ, Walker R. Cytomorphologic Analysis of Papanicolaou Stained </p><p>Smears Collected from Floor of the Mouth Mucosa in Patients with or without Oral</p><p>Malignancy. Turk J Med Sci 2001; 31:225-228.</p><p>9 SIGNATURE OF CANDIDATE</p><p>10 REMARKS OF THE GUIDE This study will help in evaluation of better staining method for micronuclei in potentially malignant lesions of the oral cavity.</p><p>11 NAME AND DESIGNATION OF Dr. B.R. AHMED MUJIB. (IN BLOCK LETTERS) PROFESSOR & HEAD, DEPARTMENT OF ORAL PATHOLOGY 11.1 GUIDE AND MICROBIOLOGY, BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE.</p><p>11.2 SIGNATURE</p><p>11.5 HEAD OF DEPARTMENT Dr. B.R. AHMED MUJIB. PROFESSOR & HEAD, DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY, BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE.</p><p>11.6 SIGNATURE</p><p>12 12.1 REMARKS OF THE CHAIRMAN AND PRINCIPAL</p><p>12.2 SIGNATURE </p>