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. V N RATNA KUMARI T
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 /
28-05-2012
5. / TITLE OF THE TOPIC: / “ORAL CYTOSMEARS ASSESSED MORPHOLOGICALLY USING DIFFERENT STAINS (PAP, H&E AND GIEMSA) BEFORE AND AFTER APPLICATION OF TOLUIDINE BLUE IN SMOKERS AND NONSMOKERS.”
6. / BRIEF RESUME OF INTENDED WORK:
6.1 Need for study:
Oral health is essential in improving one’s quality of life. Any abnormality ranging from dental decay to fatal oral cancer affects individual’s well being. Oral cancer is a significant threat to the public health as is often not diagnosed until it is advanced.1
Tobacco usage has become a major health problem. While there are many forms of tobacco product, most common is the manufactured cigarette. Cigarette pyrolysis produces smoke containing combustion by products and free radicals which are carcinogenic.2 They act by disruption of normal cellular and molecular mechanisms.
Despite advances in detection and treatment of oral cancer, the overall survival rate is less than 50%.1 Oral cancer when detected at an early stage is often curable, inexpensive to treat and affords a better quality of life.3
Various methods have been developed to supplement clinical examination and to improve diagnosis of oral cancer in its early stage. The most evaluated adjunct for lesion detection is toluidine blue, a metachromatic acidophilic dye. Dysplasia in premalignant lesions contains much more DNA and RNA than the normal epithelium due to active cellular proliferation. So use of in vivo staining by means of toluidine blue is based on the fact that it selectively stains acidic tissue components such as DNA and RNA.4 Oral lesions stained with toluidine blue showed consistent loss of chromosomal genetic information, termed loss of heterozygosity.
Previous studies showed that sensitivity and specificity of toluidine blue were quite reliable in revealing early oral premalignant lesions and monitoring recurrences of oral cancer.5,6 Toluidine blue positive staining correlated with clinicopathologic risk factors and high risk molecular patterns.7
A question needs to be answered is whether toluidine blue can be used to screen premalignant lesions occurring in smokers.
Cytological study of oral cells is a non invasive technique that is well accepted by the patient, and therefore a better option for early diagnosis of potentially malignant lesions in smokers.1% toluidine blue is used to detect dysplasia and cytological changes in oral smears.
Many studies with toluidine blue used Papanicoloau kit for staining smears. Studies carried out showed that toluidine blue enhanced the staining characters of pap stain by improving cytological features and stain quality.2 However little attention has been given to the effect of different staining procedures in toluidine blue application.
Aim of the present study is to assess the smears before and after application of toluidine blue in smokers against non-smokers and also study the effects of different stains( PAP, H E, GIEMSA) in assessing the smears.
Research hypothesis(H1):
There is a difference in the cytological features of smears of smokers against non smokers before and after application of toluidine blue and also there is difference in staining (Pap, H&E and Giemsa) of smears.
Null hypothesis(H0):
There is no difference in the cytological features of smears of smokers against non smokers before and after application of toluidine blue and also no difference in staining (Pap, H&E and Giemsa) of smears.
6.2 Review of literature:
A study was conducted on the morphological assessment of smears before and after application of toluidine blue in smokers and non smokers. It assessed cytological changes using PAP stain. This study showed that toluidine blue enhanced the staining characteristics of PAP in terms of specificity (clinical site delineation) and sensitivity (improved stain quality and enhanced cytological features). This study concluded that use of toluidine blue is synergistic in assessment of cytological smears with PAP stain.2
A study conducted determined the usefulness of toluidine blue staining as a diagnostic tool for precancerous and cancerous oral cavity lesions. This study used toluidine blue as a marker to differentiate lesions at high risk of progression in order to improve early diagnosis of oropharyngeal carcinoma. In conclusion toluidine blue stain has been shown to be a reliable aid when clinical examination fails to differentiate lesions at high risk of progression and thus improves early diagnosis for oral cavity and oropharyngeal cancer.4
A study was done on cytomorphometric analysis of the buccal mucosa of tobacco users. This study assessed the effect of tobacco smoking and of betel quid chewing with tobacco on buccal mucosa by cytomorphometry. Cellular diameter and nuclear diameter of exfoliated buccal squames obtained from clinically normal appearing buccal mucosa of tobacco smokers, tobacco chewers and those with combined habit, stained by Papanicolaou method were measured. A statistically significant reduction in cellular diameter and increase in nuclear diameter in smokers and those with combined habit were observed. So study concluded that the use of tobacco in the form of smoking influenced the cytomorphology of buccal mucosa.8
One of the studies evaluated staining methods for cytological diagnosis of oral lesions and compared efficacy of PAP, H/E, LEISHMAN and PAS. Different stains were compared regarding quality of definition of cytoplasmic and nuclear morphological characteristics and identification of bacteria, fungi, inflammatory cells and secretions. Pap and H and E were considered better methods for cytological diagnosis. Study concluded that cytological diagnosis of oral lesions along with different stains is a useful tool for oral diagnosis.9
A study was done which comparatively analyzed toluidine blue with frozen sections in oral squamous cell carcinoma. This study showed that toluidine blue can be used as an effective screening modality for assessment of intra operative margins in resource limited environment and reducing the number of frozen section biopsies performed. Further by providing clinical information within minutes it reduced indirect costs such as operating room time.10
6.3 Objectives of the study:
1.  To assess the cytological changes in smokers against non-smokers before and after application of toluidine blue.
2.  Evaluate the efficacy of different stains, Papanicolaou, Haematoxylin and Eosin, and Giemsa in assessing oral smears.
7. MATERIALS AND METHODS:
7.1 Source of data:
Study sample will include 50 individuals (25 smokers and 25 non smokers) with no clinical apparent lesions, reporting to the Department of Oral Medicine and Radiology and Department of Oral Pathology and Microbiology, at Bapuji Dental College and Hospital, in Davangere.
Estimation of sample size:
Based on the information available from previous studies, sample size was determined by using the formula and table of sample size.
Cohen’s delta = Expected mean difference
Average standard deviation
Level of significance= 5%
Power of the study= 80% = (1−β)
By taking the most significant values from key article, expected mean difference was 5.29 and average standard deviation was 6.13.By substituting the above values, Cohen’s delta(d) came around 0.86, taken as 0.9. For this value, the minimum sample size was found to be 21 by referring the table of sample size. So 25 subjects will be studied in each group.
7.2 Method of collecting data (including sampling procedures, if any):
Study sample will include oral cytological smear from smokers without clinically apparent lesions. Smoking history regarding duration of smoking and number of cigarettes smoked per day will be obtained from each smoker. Healthy subjects with clinically normal oral mucosa will be taken as controls. The written consent, to carry out in vivo staining with toluidine blue and to take cytological smears, for the study will be obtained after the necessary instructions. Ethical approval certificate has been obtained from Institutional Review Board.
INCLUSION CRITERIA:
Subjects included in the study will be smokers with minimum smoking history of 6 months and without clinically apparent lesions. All of them are males aged 18yrs or more.
EXCLUSION CRITERIA:
Patients with provisional/confirmed diagnosis of any cancer and with any clinical apparent lesions will not be included in the study. Patients with diabetes, heart and neurological diseases will also be excluded.
Study sample will be divided into two groups:
Group1: Control group comprise of 25 (non smokers) healthy volunteers with clinically normal oral mucosa.
Group2: Study group comprise of 25 smokers without clinically apparent lesions.
Three cytosmears from one side of the buccal mucosa of subjects, will be obtained before toluidine blue application and three after the application, from the same side. Subjects will be instructed to rinse the oral cavity with water for 20 seconds and then three smears will be taken using cytobrush. Subjects then will be asked to rinse with 10ml of 1% acetic acid for 30seconds followed by 1% toluidine blue application for 20sec and finally asked to rinse with 1% acetic acid. Then three more smears will be taken, in each subject. All the smears will be transferred to dry glass slides and fixed with microanatomy fixative. Smears then will be stained separately using the following stains:
1.  Papanicolaou stain
2.  Haematoxylin and eosin stain
3.  Giemsa stain
The smears will then be assessed and observed under 40x and 100x magnifications. An eyepiece grid will be used and 100 cells per slide will be counted. The whole slide is scanned in a zigzag manner from one end to other end to note the changes.
Cytological features in the smears will be grouped as
a)  Architectural changes; agglomeration or clumping of squamous cells.
b)  Cellular changes; cellular pleomorphism and presence of micronuclei.
c)  Nuclear changes; binucleation
d)  Other changes; includes presence of bacterial colony units and keratin flakes.
Statistical analysis:
Results will be subjected for appropriate statistical analysis. Group wise comparisons will be made by unpaired `t’ test/Mann whitney test depending on type of data generated. Comparison between stains is done using ANOVA 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, in vivo toluidine blue application and oral cytosmears will be carried on study cases, who smoke (without clinically apparent lesions) and subjects with clinically normal oral mucosa to be taken as controls.
7.4: Has ethical clearance been obtained from your institution in case of 7.3.
Yes.
8. LIST OF REFERENCES:
1.  Chen YW, Lin JS, Wu CH, Lui MT, Kao SY, Fong Y. Application of in vivo stain of methylene blue as a diagnostic aid in the early detection and screening of oral squamous cell carcinoma and precancerous lesions. J Chin Med Assoc 2007 Nov;70(11).
2.  Yerlagudda K, Kamath VV, Satelur K. Morphological assessment of oral cytological smears before and after application of toluidine blue in smokers and non-smokers. International Journal of Oral &Maxillofacial Pathology 2012;3(1):08-14.
3.  Nagaraju K, Prasad S, Ashok L. Diagnostic efficiency of toluidine blue with lugol’s iodine in oral premalignant and malignant lesions. Indian J Dent Res 2010;21(2).
4.  Allegra E, Lombardo N, Puzzo L, Garozzo A. The usefulness of toluidine staining as a diagnostic tool for precancerous and cancerous oropharyngeal and oral cavity lesions. Acta Otorhinolaryngol Ital 2009;29:187-90.
5.  Onofre MA, Spoto MR, Navarro CM. Reliability of toluidine blue application in the detection of oral epithelial dysplasia and in situ and invasive squamous cell carcinomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:535-40.
6.  Zhang L, Williams M, Poh CF, Laronde D, Epstein JB, Durham S et al. Toluidine blue staining identifies high risk primary oral premalignant lesions with poor outcome. Cancer Res 2005;65:8017-21.
7.  Epstein JB, Zhang L, Poh C, Nakamura H, Berean K, Rosin M. Increased allelic loss in toluidine blue positive oral malignant lesions. Oral Surg Oral Med Oral pathol Oral Radiol Endod 2003;95:45-50.
8.  Einstein TB, Sivapathasundharam B. Cytomorphometric analysis of the buccal mucosa of tobacco users. Indian J Dent Res 2005 Apr-June;16(2):42-6.
9. Almeida JD, Lima CF, Brandao AA, Cabral G. Evaluation of staining methods for cytologic diagnosis of oral lesions. Acta Cytol 2008;52:697-701.
10. Junaid M, Chaudhari MM, Sobani ZA, Murtaza G, Qadeer S, Ali NS et al. A comparative analysis of toluidine blue with frozen section in oral squamous cell carcinoma. World J Surg Oncol 2012 Apr 16;10:57.

BAPUJI DENTAL COLLEGE AND HOSPITAL, DAVANGERE

DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY

INFORMED CONSENT

I ______the undersigned hereby give my consent for the investigation being carried upon me. I am satisfied with the information given about this clinical study titled “ORAL CYTOSMEARS ASSESSED MORPHOLOGICALLY USING DIFFERENT STAINS BEFORE AND AFTER APPLICATION OF TOLUIDINE BLUE IN SMOKERS AND NONSMOKERS” being conducted by Dr. V N RATNA KUMARI T under the guidance of Dr. AHMED MUJIB B.R. MDS., Professor & Head of Department. I have been informed and explained the risk involved and I hereby voluntarily and unconditionally give my consent without any fear or pressure, in mentally sound and conscious state to participate in this study.

Date: Patient’s Signature

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