Rajiv Gandhi University of Health Sciences s190
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. / Name of the Candidate And Address(in block letters) / Dr. SONAL GROVER
POST GRADUATE STUDENT,
DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE-577004, KARNATAKA.
2. /
Name of the Institution
/ BAPUJI DENTAL COLLEGE AND HOSPITAL,DAVANGERE-577004, KARNATAKA.
3. / Course of Study
AND SUBJECT / MASTER OF DENTAL SURGERY
ORAL PATHOLOGY AND MICROBIOLOGY.
4. / Date of admission to THE COURSE / 21/04/20095. /
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.
7. / BRIEF RESUME OF INTENDED WORK:
6.1 Need for the study
“Prevention is better than cure"Many oral cancers are preceded by clinically evident premalignant mucosal changes that give a warning of risk and present an opportunity for detection and preventive measures. The key to diagnosis is the early detection of mucosal changes that may represent disease and not variations of normal. Cytological study of oral cells is a nonaggressive technique that is well accepted by the patient, and is therefore an attractive option for the early diagnosis of potentially malignant lesions.1
The analysis of micronuclei (MN) has gained increasing popularity as an in vitro genotoxicity test and a biomarker assay for human genotoxic exposure and effect. In comparison with chromosomal aberrations (CA), the scoring of MN is simpler, requires shorter training and is less time consuming. In principle, the MN assay can be expected to be more sensitive than the CA assay, because of the increased statistical power brought about by the fact that the number of cells analysed can easily be increased to thousands when only a hundred or a few hundred cells are usually scored for chromosomal aberrations.2
Micronucleus (MN) is defined as microscopically visible, round or oval cytoplasmic chromatin mass next to the nucleus. Micronuclei originate from aberrant mitoses and consist of eccentric chromosomes, chromatid fragments or whole chromosomes that have failed to be incorporated into the daughter nuclei during mitosis.3
It is well established that potentially malignant lesions have got significant number of micronuclei which can be used in high risk population as a screening test. Many studies with epithelial cells have been published in the last twenty five years. However, only little attention has been, until now, given to the effect of different staining procedures on the results of micronuclei assays. Majority of the studies on exfoliated oral mucosal cells are conducted with Papanicolaou stain. Nuclear anomalies (and possibly keratin bodies) may be misinterpreted as micronuclei with non-specific DNA stains and lead to false positive results.1, 4
The aim of the present study is to investigate if, and to what extent, different stains have an effect on the results of micronuclei in exfoliated oral mucosal cells. Oral exfoliated cells from the potentially malignant lesions will be taken and frequencies of micronuclei will be compared using three different stains, i.e. Feulgen stain, Papanicolaou stain and Haemotoxylin & Eosin stain.
6.2 Review of literature:
1. In Medical University of Vienna, Austria, a study was conducted on the effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosal cells using two DNA non-specific stains (May-Grunwald-Giemsa and Giemsa) and three DNA specific stains (Feulgen, Acridine orange, and 4’,6-diamidino-2-phenylindole (DAPI). Results showed with Giemsa based stains, the frequencies of micronuclei in smokers were significantly (4 to 5 times) higher than in non-smokers group whereas, no significant increase was observed with any of the DNA specific stains.4
2. In a study carried out at National Institute for research on cancer, Genoa, Italy, the influence of methodological variables, staining method and sampling, on the frequency of micronuclei scored in squamous epithelial cells of oral mucosa were studied. Feulgen stain, Giemsa stain and fluorescent dyes (Hoechst 33258 and Propidium iodide) were used for micronucleus staining. Results showed that staining of micronuclei by Hoechst 33258 proved to be more handy, less time consuming and equally reliable as compared to the Feulgen staining. Results with Giemsa stain and Propidium iodide were unsatisfactory.5
3. In a study, using Feulgen stain for staining the micronuclei, it was summarised that defining and using a strict protocol is a prerequisite for counting micronuclei in exfoliated cells to get a reproducible and sensitive indicator of cancer risk.6
4. A study conducted in Ghazi University, Ankara-Turkey, concluded that cytomorphologic assessment of the Papanicolaou stained oral smears collected from malignant and premalignant lesions in the floor of the mouth could provide an additional diagnostic test for monitoring such lesions and thus detecting oral malignancy at an early stage.7
6.3 Objectives of the Study:
1. To study the selectivity of micronuclei in exfoliated oral mucosal cells with Feulgen stain, Papanicolaou stain and Haemotoxylin & Eosin stain in potentially malignant lesions.
2. To evaluate the frequency of micronuclei in patients with potentially malignant lesions and comparison with healthy subjects as controls.
MATERIALS AND METHODS:
7.1 Source of Data:
Study sample will include subjects reporting to the Department of Oral Medicine and Radiology and Oral Pathology and Microbiology, Bapuji Dental College and Hospital, Davangere as outpatients with lesions clinically diagnosed to be potentially malignant.
7.2 Method of Collecting Data (Including Sampling Procedures, If Any):
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.
INCLUSION CRITERIA:
Subjects included in the study will be those clinically diagnosed as one of the following potentially malignant lesions of the oral cavity:
Leukoplakia, Erythroplakia, Oral submucous fibrosis and Lichen planus.
EXCLUSION CRITERIA:
Patients with provisional or confirmed diagnosis of any cancer will not be included in the study.
Study sample will be divided into two groups:
Group 1: Control group comprise of 10 healthy subjects with clinically normal oral mucosa.
Group 2: Comprise of 40 patients with clinically diagnosed as potentially malignant lesions of the oral cavity.
Three cytosmears from lesions of the subjects will be obtained as follows:
The subjects will be asked to rinse their mouth with water and a cytobrush will be used to obtain exfoliated cells from the oral mucosa. The samples will be transferred to three dry glass slides, to ensure an adequate harvest of cells. Smears will be air dried and fixed with 95% ethanol spray. Smears will be separately stained with following stains:
1. Feulgen stain
2. Papanicolaou stain
3. Haemotoxylin & Eosin stain.
For designating an extra nuclear body as micronucleus, the following criteria given by Tolbert3 will be considered and 1000 cells will be scored to determine the micronuclei frequency:
(a) Rounded smooth perimeter suggestive of a membrane.
(b) Less than a third the diameter of the associated nucleus, but large enough to discern shape and color.
(c) Staining intensity similar to that of the nucleus.
(d) Texture similar to that of nucleus.
(e) Same focal plane as nucleus.
(f) Absence of overlap with, or bridge to, the nucleus.
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-Whitney U test).
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.
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.
7.4: Has ethical clearance been obtained from your institution in case of 7.3.
Yes
List of References:
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.
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.
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.
5. Casartelli G, Monteghirfo S, De Ferrari M, Bonatti S, Scala M, Toma S et al . Staining of micronuclei in squamous epithelial cells of human oral mucosa. Anal Quant Cytol Histol 1997; 19(6):475-81.
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.
7. Mollaoglu N, Cowpe GJ, Walker R. Cytomorphologic Analysis of Papanicolaou Stained
Smears Collected from Floor of the Mouth Mucosa in Patients with or without Oral Malignancy. Turk J Med Sci 2001; 31:225-228.
8.
9 / SIGNATURE OF CANDIDATE
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.
11 / NAME AND DESIGNATION OF
(IN BLOCK LETTERS)
11.1 GUIDE
11.2 SIGNATURE / Dr. B.R. AHMED MUJIB.
PROFESSOR & HEAD,
DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE.
11.5 HEAD OF DEPARTMENT
11.6 SIGNATURE / Dr. B.R. AHMED MUJIB.
PROFESSOR & HEAD,
DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE.
12 / 12.1 REMARKS OF THE CHAIRMAN AND PRINCIPAL
12.2 SIGNATURE