Cytomorphometric Study of Exfoliated Cells of Buccal Mucosa in Type II-Diabetic Patient

 

Dr. Vidya G Doddawad1, Dr. Shivananda S2Dr. Pradeep Subbaiah3

1Associate Professor, Department of Oral Pathology and Microbiology, JSS Dental College and Hospital A Constituent College of JSSAHER Mysore-570022

2 Associate Professor, Department of Oral and Maxillofacial Surgery, JSS Dental College and Hospital A Constituent College of JSSAHER Mysore-570022

3Senior Lecturer, Department of Orthodontics JSS Dental College and Hospital A Constituent College of JSSAHER Mysore-570022

*Corresponding Author E-mail: drvidyagd@gmail.com

 

ABSTRACT:

India is the country with highest prevalence of diabetes mellitus in the world. Other condition like premalignant lesions and condition, oral exfoliative cytology may be more appropriate as the non-invasive techniques for early diagnosis. So, cytology technique may help to diagnose diabetes disease and help to prevent the complications. Aims: The present study is conducted to evaluate the cytological and cytomorphometric changes of exfoliated cells of buccal mucosa in type-II diabetic patients. Methods and Material: The diabetic group comprised of 30 diabetic patients, the age of 30-60 years suffering from diabetes mellitus at least from one year. Control group comprised of 15 healthy volunteers above the age of 30 years. Smears were taken from the buccal mucosa and stained with papanicolaou stain. The stained smears were measured for the cellular diameter (CD) and nuclear diameter (ND) along the longest axis of the cell using software digital camera with axiovision of version 2.0. Collected data were analyzed using ‘Unpaired student t test’. Results: The results showed the mean nuclear diameter of the exfoliated cells of diabetic group was 11.198μm and that of the control group was 9.494μm and the difference showed very highly significance (P<0.001). The mean cell diameter of the exfoliated cells of diabetic group showed 56.52μm and the control group showed 53.148μm and the difference between these group was significant (P<0.01). Conclusions: This study suggests that there were significant cellular and nuclear changes in the diabetic patients. The knowledge of which can be helpful for the cytopathologist in the early recognition of the cellular changes in the diabetic patients.

 

KEYWORDS: Diabetes mellitus; Cytomorphometry; Oral mucosa; Buccal mucosa; Type II.

 

 


INTRODUCTION:

Diabetes mellitus is a massively growing epidemic disease that has requires the potential health care services in the world. India is one of the countries with highest prevalence of diabetes mellitus. It is basically characterized by chronic hyperglycemia, associated with disturbances in the metabolism of carbohydrates, proteins and lipids, resulting in insulin deficiency.

 

 

Chronic hyperglycemia causes damage to the eyes, kidneys, nerves, heart and blood vessels. The etiology and pathophysiology leading to the hyperglycemia, however, are markedly different patients with diabetes mellitus, dictating different prevention strategies, diagnostic screening methods and treatments1. Early diagnosis of this disease will help to prevent the complications.

 

Sometimes diabetes often undergoes undiagnosed because of its symptomless features. Therefore, to monitoring of glycosylated hemoglobin (GHb) levels in the blood has become much commoner and it is invasive method 2. To avoid this, there is oral exfoliative cytology has taken more interest which is noninvasive diagnostic procedure2 and dentist helps in the early diagnosis of this disease as this is associated with various oral manifestations. Hence, the present study was conducted on the cellular changes like cellular diameter and nuclear diameter of the exfoliated cells of the buccal mucosa in diabetic patients and comparison with the control group.

 

MATERIAL AND METHODS:

The present study was conducted in the diabetic clinic and the outpatient department of our institution which consisted of thirty diabetic patients (case group) above the age of thirty years suffering from diabetes mellitus for at least one year and their present fasting whole venous blood sugar level (> 110mg dl-1) were included. Fifteen healthy non-diabetic people (Control group) above the age of thirty years formed the control group. Patients with known local and systemic disease, medically compromised patients, patients with habits (Pan chewing, Alcohol abuse and Smoking), abnormal endocrinal and immunological status, anemia and ulcerative conditions, and frank oral local lesions other than gingivitis, were excluded from the study and control group.

 

Informed consent was obtained for both diabetic (case) group and Non diabetic (Control) group and a data sheet was recorded detailed case history like name, age, sex, relevant medical history, oral habit history etc. The smears were taken from both case and control group. Smear were obtained from healthy buccal mucosa adjacent to the first and second maxillary molars, above the line of occlusion by using Oral B interdental brush. (Fig.1) The scrapped material were smeared over the plain glass slide which was specifically coded for each individual with diamond pencil. The smears were immediately fixed in absolute alcohol for 30 min, and then stained with Papanicolaou stain. Stained smears were observed under a binocular bright-field microscope using software Axiovision 2.0. (Fig.2)

 

The slide was read from left to right then down and across in a Zigzag manner to avoid measuring the same cells repeatedly. Clumped, folded cell and distorted cells were not considered and fifty cells were measured for the nuclear diameter (ND) and cell diameter (CD). The obtained nuclear diameter and cell diameter were recorded and calculated. The data were then analyzed using statistical method “Unpaired student t test”. Reliability of intra and inter observer bias was calculated.

 

RESULTS:

The results of the present study showed the mean nuclear diameter of the exfoliated cells of buccal mucosa of diabetic group was 11.198 ± 0.62370μm and that of the control group was 9.494 ± 0.48246 μm (Table.1). The mean cell diameter of the exfoliated cells of buccal mucosa in diabetic group showed 56.52 ± 3.73675 μm and the control group showed 53.148 ± 3.97918 μm (Table. 2). The difference in nuclear diameter of the Diabetic group and control group is very highly significant (P<0.001) (Graph.1). The difference in the cell diameter of the Diabetic group and control group is highly Significant (P<0.01) (Graph.2) (Table.3)

 

DISCUSSION:

Diabetes mellitus is a complex multisystem disorder which is associated with various oral manifestations because of which it is more commonly encountered by the dentist. Hence dentist plays very important role in the early diagnosis of the disease. Early diagnosis is helpful in the control of blood sugar level at a very early stage and to prevent various complications. In recent years important advances have occurred in the determination of diagnostic criteria for the disease diabetes mellitus and also in new strategies for its treatment.

 

Exfoliative cytology is the simple and non invasive technique which is mainly used as screening test of oral squamous cell carcinoma. Because of the renewed interest in the potential role of oral exfoliative cytology it has lead its applications in the diagnosis of many endocrinal disturbances.

 

In this study, cytology is applied to evaluate the cellular and morphometric changes in the exfoliated cells of the buccal mucosa of 30 diabetic patients suffering from the disease for at least one year and compared with the exfoliated cells of the buccal mucosa of 15 healthy people. Diabetic patients, above the age of 30 years and with fasting blood sugar level above 110 mgdl-1, following WHO diagnostic criteria 19991, were chosen for the study.

 

Most of the cellular and nuclear features of the exfoliated cells of the oral mucosa of the diabetic patients were similar to the features of the healthy mucosa. However there were certain features observed in the diabetic groups. Smears of diabetic patients showed abnormal cellular changes like karyorrhexis, binucleation and micronucleus. This finding is in consistency with the study done by Alberti et al3.

 

The results of the present study showed the mean nuclear diameter of the exfoliated cells of buccal mucosa of diabetic group was 11.198±0.62370μm and that of the control group was 9.494±0.48246 μm. On statistical analysis, the difference in nuclear diameter between diabetic group and control group showed very highly significant (P<0.001). This is in consistent with the study done by Alberti et.al3 according to whom the nuclear area was markedly higher in diabetic group (P<0.05). The differences in nuclear area and diameter exist between basal and spinous cells during epithelial differentiation and maturation in normal epithelium also. But significant increase in nuclear area and diameters are presented in neoplastic lesions3. Similar study was conducted by Maeda MYS et al4 stated that there is significant increase in nuclear diameter, uniformity in nuclear configuration supports a benign change in the cellular pattern. The possible hypothesis for explaining the increase in mean ND by Manish et.al, an increased glucose level directly influence on the cell growth because of its pivotal role in metabolic processes. An actively growing cell is characterized by a prominent and large nucleus5.

 

The mean cell diameter (CD) of the exfoliated cells of buccal mucosa in diabetic group showed 56.52±3.73675 μm and the control group showed 53.148 ± 3.97918 μm. On statistical analysis the difference in cellular diameter between diabetic group and control group showed high significant value (P<0.01). This finding is matching with the findings of Jajarm et al. who reported an increase in cytoplasmic area in diabetic patients6. This did not match the finding of Alberti et al3 according to their study cytoplasmic area of the diabetic group did not exhibit statistically significant difference (P>0.05).

 

The quantitative cellular alterations like significant increase in the nuclear diameter found in the exfoliated cells of the buccal mucosa of the diabetic patients may be seen in various other conditions like smokers, nutritional deficiency like hypochromic anaemia, megaloblastic anaemia, Vit B12 and Folic acid deficiency.3 Therefore the present study, we exclude these patients with these condition which may affect the results of the study.

 

Instruments like metal spatula, cotton sticks, wooden spatula and brush are used for adequate sample collection. According to Jones AC et al7 cytobrush obtained adequate number of cells and cells were dispersed in a thin uniform layer. In this study we used interproximal brush for sample collection.

 

Various morphometric methods are used by different authors for different cytomorphometric studies. According to Dandona et al8 poor diabetic control is associated with an increased cancer risk due to enhanced oxidative damage to DNA. Seppala B et al9

 

Described that diminished immunity in diabetic patients and microangiopathy which causes hypoxia and a reduced blood supply together may play a role in development of oral cancer. Tsuji S et al10 and Seril DN et al11 stated that alterations occurred in the oxidative equilibrium of free radicals in diabetic patients. Elevated blood glucose levels can lead to excessive formation of free radicals. Also due to protein breakdown, the activity of antioxidant scavengers and enzymes is reduced. Both the increase in free radicals and oxidative stress promote carcinogenesis. Thus even though the oral mucosa of diabetic patients appears clinically normal, microscopically cytomorphometric changes are noted which explain the result in our study.

 

In the background of the association of diabetes mellitus with various neoplastic and inflammatory diseases the early changes in the oral cavity can be ascertained through cytology more so through cytomorphometry. But still elaborate studies are required to show significant association.

 

CONCLUSION:

From the present study, we conclude that diabetes mellitus patients shows definite morphological and morphometric changes in the exfoliated buccal mucosal cells. The change in nuclear area and cellular area is very much significant. However, for establishing exfoliative cytology as an additional diagnostic noninvasive tool for evaluation of diabetes and further studies is required with a larger sample size to establish a deeper insight effect on the oral and other tissue.

 

REFERENCE:

1.     Alberti KG, Zimmet PZ. Definition , diagnosis and classification of Diabetes mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus Report of a WHO consultation. Geneva: World Health Organisation; 1999. (Department of Noncommunicable Disease Surveillance).

2.     Williams G, Pickup JC. Handbook of diabetes. 3rd ed. London: Blackwell Publishing; 2004. pp. 56–62.

3.     Alberti S, Spadella CT, Francischone TRCG, Assis GF, Cestari TM, Taveira LAA. Exfoliative cytology of the oral mucosa in type II diabetic patients: morphology and cytomorphometry. J Oral Pathol Med 2003; 32: 538-43.

4.     Maeda MYS, Loreto CD, Shirata NK, Shih LWS, Cavaliere MJ, Filho AL et al. Image analysis of Nuclear / Cytoplasmic Ratio in Cervical smears to discriminate three grades of Cervical Intraepithelial Neoplasia. Acta Cytologica 1997; 41: 744- 748.

5.     Manisha S, Hema S, Sushruth N, Pramod K. Cytomorphometric analysis of gingival epithelium and buccal mucos cells in type2 diabetes mellitus patients. JOMFP 2017, 21(2):224-228.

6.     Jajarm HH, Mohtasham N, Moshaverinia M, Rangiani A. Evaluation of oral mucosa epithelium in type II diabetic patients by an exfoliative cytology method. J Oral Sci. 2008;3:335–40.

7.     Jones AC , Pink FE, Sandow PL, Stewart CM, Migliorati CA, Baughman RA et al. The cytobrush plus cell collector in oral cytology. Oral Surg Oral Med Oral Pathol 1994;77:101-4.

8.     Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D. Oxidative damage to DNA in diabetes mellitus. Lancet 1996; 347:444–5.

9.     Seppala B, Sorsa T, Ainamo J. Morphometric analysis of cellular and vascular changes in gingival connective tissue in long-term insulin-dependent diabetes. J Periodontol 1997; 68(12):1237–45.

10.     Tsuji S, Kawai N, Tsujii M, Kawano S, Hori M. Review article: inflammation related promotion of gastrointestinal carcinogenesis-a perigenetic pathway. Ailment Pharmacol Ther 2003; 18:82–9.

11.     Seril DN, Liao J, Yang GY, Yang CS. Oxidative stress and ulcerative colitis- associated carcinogenesis: studies in humans and animal models. Carcinogenesis 2003; 24(3):353–62.

 

 

 

 

 

 

 

 

 

Received on 22.11.2018         Modified on 10.12.2018

Accepted on 26.12.2018      © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(3): 1178-1180.

DOI: 10.5958/0974-360X.2019.00194.X