DISSERTATION – SYNOPSIS



DISSERTATION – SYNOPSIS

Dr. SMIT SINGLA

Post graduate student

Department of Oral Medicine and Radiology

A .J. INSTITUTE OF DENTAL SCIENCES, MANGALORE

2009 – 2010

Rajiv Gandhi University of Health Sciences, Karnataka.

Bangalore

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

| |Name of the Candidates and Address |Dr. SMIT SINGLA |

|1. | |POST GRADUATE STUDENT |

| | |DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY |

| | |A.J. INSTITUTE OF DENTAL SCIENCES, MANGALORE |

|2. |Name of the institution | A.J. INSTITUTE OF DENTAL SCIENCES, MANGALORE |

|3. |Course of study and subject |Master of Dental Surgery |

| | |ORAL MEDICINE AND RADIOLOGY |

|4. |Date of admission to course |20 May 2009 |

| |Title of the topic: |

|5. |MICRONUCLEUS ASSAY – AN EARLY DIAGNOSTIC TOOL TO ASSESS GENOTOXIC CHANGES IN TOBACCO AND RELATED HABITS |

| |BRIEF RESUME OF THE INTENDED WORK |

| |6.1 Need for the study |

|6. |Oral cancer is by far the most common oral mucosal malignant disease. Though the diagnosis of oral cancer seldom presents difficulty, |

| |it is the early detection, cancer staging and histological grading that are more important for prognosis. |

| |As documented the phenotypic changes of oral cancer is always preceded by genetic damages accumulated due to various carcinogens. It is|

| |the need of the hour, to detect these genetic fingerprinting at the earliest, even before dysplastic changes is attributed to the cell.|

| | |

| |Among the various non-invasive early detection tools, the use of Micronuclei (MN) as a marker for the above effect has been well |

| |documented and has a sensitivity and specificity of 94% and 100% respectively1. |

| |Micronuclei are induced in cells by a variety of substances, like ultraviolet radiation, infrared rays, X-radiations, and chemicals. |

| |Among them tobacco- specific nitrosamines have been reported to be potent mutagenic agents which are thought to be responsible for the |

| |induction of chromosomal aberrations resulting in production of micronuclei. |

| |As documented the effects of smokeless tobacco and tobacco smoking is varied. Hence an appropriate tool for early detection of tobacco |

| |related genotoxic effects on a cell even before phenotypic changes of cancer are evident1. |

| |Micronuclei are extranuclear cytoplasmic bodies, caused due to double strand chromosomal aberrations and detected through appropriate |

| |staining procedure. These damaged chromosomes, in the form of acentric chromatids or chromosome fragments, lag behind in anaphase when |

| |centric elements move towards the spindle poles. The lagging elements are included in the daughter cells too, but a considerable |

| |portion is transformed into one or several secondary nuclei, which are as a rule, much smaller than the principal nucleus and are |

| |therefore called micronuclei2, 3. |

| |The detection of such micronuclei can be done in various types of tissues from the body (RBC, reticulocytes, lymphocytes and exfoliated|

| |epithelial cells). The exfoliated cell Micronucleus assay has an advantage over the widely used Micronucleus test in lymphocytes |

| |because, an epithelial cell unlike a lymphocyte, do not need to be stimulated to undergo mitosis since they are a constantly dividing |

| |pool of cells4. The use of buccal exfoliated cells in the detection of genotoxic effects of tobacco and related products can be used as|

| |a mass screening procedure for the early detection of dysplastic changes and oral cancer. |

| |6.2 REVIEW OF LITERATURE : |

| |Paige E. Tolbert, Carl M. Shy and James W. Allen (1991)4 established a revised protocol for the exfoliated cell micronucleus assay was |

| |field-tested in a population exposed to a genotoxic agent, snuff, at level associated with a significant increase in cancer risk. The |

| |standard assay involves examination of epithelial smears to determine the prevalence of micronucleated cells, an indication of |

| |chromosome breakage or mitotic interference. |

| |Paige E. Tolbert, Carl M. Shy and James W. Allen(1992)3 evaluated the exfoliated cell micronucleus assay involving microscopic |

| |analysis of epithelial smears to determine the prevalence of micronucleation, an indicator of structural or numerical chromosome |

| |aberrations. Refinement in micronucleus scoring criteria and the inclusion of other nuclear anomalies in the scoring system were |

| |proposed. |

| |Devendra Devendre H Palve, Jagdish V Tupkari.(2008)2 investigated that micronuclei were good prognostic indicators in oral Squamous |

| |cell carcinoma. Micronucleus frequencies in oral exfoliated cells stained with papanicolaou stain were counted and correlated with the |

| |histopathological grades and clinical stages of Squamous cell carcinoma patients. They were also compared with healthy control |

| |subjects. Micronuclei (MN) frequencies were also found higher in Squamous cell carcinoma patients than in control subjects. MN |

| |frequencies were also found to be raised with increasing histological grades of Squamous cell carcinoma. |

| |Sudha Sellappa, Mythili Balakrishnan, Sangeetha Raman and Subashini (2009)1 evaluation of micronuclei in buccal mucosa of healthy |

| |individuals from southern India, who were regularly chewing a mixture of betel leaf, areca nut and tobacco. The mean percentage of MN |

| |was 1.90±1.03 in chewers, 2.00±1.12 in chewers with smoking habits and 0.81±0.66 in controls. There was no significant difference |

| |between the mean percentages of the two experimental groups. It can be concluded that a mixture of betel leaf, areca nut, and tobacco |

| |is unsafe for oral health. |

| |Beena P. Patel, Pina J. Trivedi, Manisha M. Brahmbhatt, Shilin N. Shukla, Pankaj M. Shah and Sonal R. Bakshi (2009)5 analysed the |

| |tobacco related genotoxic effects in chewers monitoring micronuclei (MN) and chromosomal aberrations. The biomarker was compared with |

| |non chewer to predict the risk of genotoxicity. |

| |. |

| |6.3 OBJECTIVES OF THE STUDY: |

| |To determine the specificity and sensitivity and consequent early detection of dysplastic changes through MN assay in tobacco and |

| |related habits. |

| |To compare MN frequency among subjects, chewing tobacco only, chewing and smoking tobacco only, and chewing, smoking with alcohol, and |

| |to co-relate with control subjects. |

|7 |MATERIAL AND METHODS : |

| |Evaluation of micronucleated cell from buccal exfoliated cell using Acridine Orange fluorescent Staining. |

| |7.1 SOURCE OF SAMPLE : |

| |Buccal smear will be made after obtaining a suitable consent form healthy patients who are visiting the department of Oral Medicine and|

| |Radiology, A.J. Institute of Dental Sciences, having habits of smoking, chewing tobacco and alcohol and those without any habit. |

| |CLINICAL INCLUSION CRITERIA: |

| |Patient should be healthy without any clinical lesion in oral cavity. |

| |Patient must have history of chewing, smoking tobacco and alcoholism. |

| |INCLUSION CRITERIA FOR TOTAL CELL COUNT: |

| |No debris. |

| |No overlap with adjacent cells. |

| |Cytoplasm intact and lying relatively flat. |

| |Nucleus normal and intact with nuclear perimeter smooth and distinct. |

| |EXCLUSION CRITERIA: |

| |Under or followed vitamins and antioxidants supplementation. |

| |Under or followed radiation therapy. |

| | |

| |7.2 METHODS OF COLLECTION OF DATA |

| |METHODOLOGY: |

| |Patient selection |

| |The sample will be selected from the healthy patients visiting to the Department of Oral Medicine and Radiology. A sought of |

| |information on inclusion and exclusion criteria obtained. Subjects will be studied in the following groups with 20 samples in each |

| |group. |

| |Group 1: chewing tobacco only. |

| |Group 2: chewing and smoking. |

| |Group 3: chewing, smoking and alcohol. |

| |Group 4: control. |

| |Collection of exfoliated cells |

| |Subjects will be asked to rinse their mouth gently with water. Mucosal cells will be scraped from buccal mucosa using a slightly |

| |moistened wooden spatula. The cells will be immediately smeared on pre-cleaned microscopic slides.3, 5 Just prior to drying; the |

| |smears were fixed with commercially available spray fixative (available as BIOFIX). Then slides will be coded to ensure observer |

| |blindness4 and will be fixed in 100% alcohol. |

| |Staining procedure |

| |ACRIDINE ORANGE SOLUTION |

| |This technique requires a pH of 6.0 for the differential staining of RNA and DNA. Formalin- fixed material does not stain |

| |satisfactorily, neither does tissue fixed in Bouin’s solution; alcohol is the fixative of choice6. |

| |0.1% aqueous acridine orange. Before use dilute one part stains with 10 part of pH 6.0 0.06M phosphate buffer to give a 0.01% solution.|

| |BUFFER |

| |pH 6.0 phosphate buffer7. |

| | |

| |DIFFERENTIATOR |

| |M calcium chloride ( 11.099g calcium chloride in 100 ml distilled water)6. |

| |Technique |

| |Take alcohol-fixed smear to distilled water. |

| |Rinse in 1% acetic acid for a few seconds and in two changes of distilled water over 1 minute. |

| |Stain in the diluted acridine orange solution at pH 6.0 for 3 minutes. |

| |Rinse in pH 6.0 buffer for 1 minutes. |

| |Differentiate in the 0.1 M calcium chloride solution for ½-1 minute. |

| |Wash in phosphate buffer and mount in the same. |

| |DNA will be stained in yellow- green. |

| |RNA, some mucins will be stained in red6. |

| |INTERPRETATION OF RESULTS: |

| |100 cells from each sample will be focused under fluorescent microscope and number of Micronucleated cells (MNC) will be counted by a |

| |single observer. |

| |7.3 Does the study requires any investigations or interventions to be conducted on patients or other humans or animals ? If So, please |

| |describe briefly. |

| |Not applicable |

| |7.4 Has ethical clearance been obtained from your institution in case of 7.3 ? |

| |Obtained |

| | |

| | |

| | |

| | |

| |INVESTIGATION DESIGN |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

| | |

|8. |LIST OF REFERENCES : |

| |Sudha Sellappa, Mythili Balakrishnan, Sangeetha Raman and Subashini. Induction of micronuclei in buccal mucosa on chewing a mixture of |

| |betel leaf, areca nut and tobacco. J. Oral Science, 2009; 51(2):289-292. |

| |Devendre H Palve, Jagdish V Tupkari. Clinico-pathological correlation of micronuclei in oral Squamous cell carcinoma by exfoliative |

| |cytology. J. Oral Maxillofac Pathol, 2008; 12:2-7. |

| |Paige E. Tolbert, Carl M. Shy and James W. Allen. Micronuclei and other nuclear anomalies in buccal smears: methods development. |

| |Mutation Research, 1992; 271:69-77. |

| |E. Tolbert, Carl M. Shy and James W. Allen. Micronuclei and other nuclear Paige anomalies in buccal smears: A field test in snuff |

| |users. American J Epidemiology 1991; 134(8): 840-850. |

| |Beena P. Patel, Pina J. Trivedi, Manisha M. Brahmbhatt, Shilin N. Shukla, Pankaj M. Shah and Sonal R. Bakshi. Micronuclei and |

| |Chromosomal aberrations in healthy tobacco chewers and control: A study from Gujarat, India. Arch Oncol, 2009; 17(1-2): 7-10. |

| |Handbook of Histopathological and Histochemical Techniques (1974). Culling, C.F.A. 3rd Edn. London; Buttterworths. (Page no. - 30) |

| |Practical Heamatology (2001).S M Lewis, B J Bain and I Bates 9th Edn. Churchill, Livingstone.(Page no.- 606) |

| | |

| | |

| | |

|9. | | |

| |Signature of Candidate | |

|10. |Remarks of the guide : | |

|11. |Name & Designation of |Dr. VATHSALA |

| |11.1 Guide : |PROFESSOR & HOD |

| | |DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY |

| | |A. J. INSTITUTE OF DENTAL SCIENCES. |

| | | |

| |11.2 Signature : | |

| |11.3 Co-Guide |Dr. RAGHAVENDRA KINI |

| | |PROFESSOR |

| | |DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY |

| | |A.J. INSTITUTE OF DENTAL SCIENCES. |

| | | |

| |11.4 Signature : | |

| |11.5 Head of Department |Dr. VATHSALA |

| | |PROFESSOR & HOD |

| | |DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY |

| | |A. J. INSTITUTE OF DENTAL SCIENCES. |

| | | |

| |11.6 Signature : | |

|12. |12.1 Remarks of the Chairman & Principal : |

| | |

| |Dr. B SURESHCHANDRA |

| |PRINCIPAL |

| | |

| | |

| |12.2 Signature |

| | |

| | |

-----------------------

80 Samples

DOUBLE BLIND STUDY

20 chewing tobacco

20 chewing & smoking

20

Chewing, smoking & alcohol

20

Control group

Buccal exfoliated cells

Acridine orange staining

Results

EXAMINATION UNDER FLUORESCENT MICROSCOPE

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download