Comparative cytomorphometric analysis of oral mucosa in patients with Diabetes, patients with associated oral habits but with apparently normal mucosa and Control group

 

Khushali K Shah¹, Dr. Gheena S²

¹II BDS, Saveetha Dental College and Hospitals, Chennai-600 077

²Reader, Department of Oral pathology, Saveetha Dental College and Hospitals, Chennai-600 077

 

ABSTRACT:

Aim: The aim of this study was to evaluate the dysplastic changes that occur in the cell, nuclear morphology and      diameter in Buccal smears of patients in the study groups.

Background: Oral exfoliative cytology is a simple, non-invasive, and painless method that is well accepted by patients and involves microscopic analysis of cells collected from the surface of oral mucosa.

Many factors affect the cytomorphology of the cells collected from oral mucosa. Some of these factors may be systemic disease; like anaemia and diabetes mellitus; radiotherapy; alcohol consumption and smoking. Cigarette and tobacco generally contain many carcinogenic substance mostly DNA-toxic carcinogens, however alcohol itself is not carcinogenic, but when combined with cigarettes or tobacco they have a worse impact on health.

 In exfoliative cytology various parameters such as nuclear size, cell and nuclear pleomorphism, nuclear membrane discontinuity, degenerative changes of nucleus and nuclear cytoplasmic ratio can be analysed.

Materials and Method: Smears were taken from the buccal mucosa of 5 patients with Diabetes, 5 patients with adverse oral habits but with apparently normal mucosa and 5 patients with normal mucosa. All the smears were stained using H & E stain and Papanicoloau stain and evaluated using research microscope and image analysis software.

Reason: To explore the potential of Exfoliative cytology as a diagnostic adjunct in patients with Diabetes and in patients with Associated habits but apparently normal mucosa.

 

 

INTRODUCTION:

Oral exfoliative cytology is a simple, non-invasive, and painless method that is well accepted by patients and involves microscopic analysis of cells collected from the surface of oral mucosa.⁽¹⁾

 

Many factors affect the cytomorphology of the cells collected from oral mucosa. Some of these factors may be systemic disease; like anaemia and diabetes mellitus; radiotherapy; alcohol consumption and smoking.

 

Cigarette and tobacco generally contain many carcinogenic substance mostly DNA-toxic carcinogens⁽²⁾, however alcohol itself is not carcinogenic, but when combined with cigarettes or tobacco they have a worse impact on health. It has been reported that substances present in cigarette smoke alter the charge and other properties of oral epithelial surfaces, allowing the growth of certain pathogenic bacteria like some species of Neisseria and certain gram-positive bacteria like Staphylococcus aureus and Streptococcus pneumonia.⁽³⁾

 

Diabetes mellitus is a complex metabolic disease which is often followed by disorders in the metabolism of carbohydrate, lipid, protein.⁽⁴⁾ Oral problems of people suffering from diabetes include xerostomia; salivary gland dysfunction; increased susceptibility to bacterial, viral and fungal infections; increase in teeth decay; inflammation of gingiva; periodontitis; periodical abscess; loss of teeth; lichen plants and burning mouth syndrome.⁽⁵⁾ There are several methods to evaluate the oral mucosa of patients with diabetes but the best method with low cost and less aggressive characteristics and also lack of damage to oral tissue of the patient is using exfoliative cytology or brush cytology.⁽⁶⁾

 

The Papanicoloau stain is regarded as the universal stain for cytological preparations since it imparts a different colour to the cytoplasm of epithelial cells based on their degree of cellular differentiation.⁽⁷⁾

 

The goal of the present study was to examine the cytomorphometric and morphometric analysis of nucleus of patients with diabetes and patients with associated habits but apparently normal mucosa and compare them to each other and to the healthy patients.

 

MATERIALS AND METHOD:

The study group consisted of 15 patients; 5 patients with diabetes, 5 patients with associated habits but with apparently normal mucosa and 5 controls.

Subjects of both the study and the control groups were informed of the procedure and a written consent was obtained.

 

Patients with diabetes:

Inclusion criteria:

Patients aged 45 years and above.

Medical history of diabetes for a minimum period of 5 years prior to commencement of the present study.

Diagnostic criteria- Random serum glucose concentration>200mg/dl, or

Fasting serum glucose level>126mg/dl.

 

Exclusion criteria:

Patients suffering from other systemic diseases.

Known cases of anaemia and malignancy.

Patients who have undergone radiation therapy and chemotherapy.

Patients with smoking or pan chewing habits and alcohol dependency.

Patients with poor oral hygiene.

Denture wearers.

 

Patients with associated habits but with apparently normal mucosa:

Inclusion criteria:

Patients aged 45 years and above.

Patients who smoked at least 10 cigarettes a day for the last 10 years, with apparently normal mucosa.

Patients with daily drinking or regular drinking for at least 10 years, with apparently normal mucosa.

 

Exclusion criteria:

Patients who suffered from systemic diseases like anaemia or diabetes.

Patients who had undergone radiation therapy or chemotherapy.

Patients with malignant or potentially pre malignant oral lesions, such as leukoplakia or erythroplakia.

Patients diagnosed with cancer.

 

Control Group:

Patients aged 45 years and above.

Patients with clinically healthy mucosa.

Patients who were non-smokers and non-alcoholics.

Patients who did not suffer from any systemic diseases.

Patients who did not have any oral lesions.

Patients with poor oral hygiene were excluded.

 

Method for collection of data:

Smear collection:

Smears were collected from clinically normal buccal mucosa of the patients using a wooden spatula moistened in distilled water. Three smears from each site were obtained. The scraping were thinly and uniformly transferred to a clean glass slide. The smears were then immediately fixed with 95% 2-propanol.

 

Two smears were stained with H&E stains and one smear was stained with Papanicolaou stain to visualise under the microscope for cytomorphometric analysis of the cells i.e., nuclear area, cellular area, cytoplasmic area and the nucleus: cytoplasm ratio.

 

The unfolded cells in a field were taken as representative of the cytological picture and evaluated. The entire slide was screened in a reister pattern for the existence of micronuclei.

 

The collected data was then compared, where the control group was compared with the diabetic group and the associated habits group, and the diabetic and associated habit groups were also compared.

 

RESULTS:

Table 1 presents the nuclear and cytoplasmic area of all the samples obtained.

NORMAL
S.NO	NUCLEAR AREA(µm2)	CELL AREA(µm2)
N1	134	3859
N2	104	2998
N3	127	3412
N4	146	4237
N5	105	2977
ADVERSE ORAL HABITS
S.NO	NUCLEAR AREA(µm2)	CELL AREA (µm2)
A1	144	4177
A2	139	4133
A3	140	4100
A4	137	3089
A5	132	3045
DIABETES
S.NO	NUCLEAR AREA(µm2)	CELL AREA (µm2)
D1	102	2735
D2	135	3617
D3	116	2583
D4	145	4819
D5	131	3173

 

Table 2: Presents the scoring as to the number of keratinized cells, number of non keratinized cells, micronuclei and the number of cells still undergoing keratinization.

 

 

Table 3: Presents the comparison of the cellular and nuclear area of normal to adverse oral habits but with apparently normal mucosa

 

 

Table 4: Presents the comparison of the cellular and nuclear area of normal to diabetes.

 

 

Table 5 presents the comparison of nuclear and cellular area of potentially malignant lesion to adverse oral habits.

 

Table 6: Presents the multiple comparison of the number of non keratinized cells among the three groups Descriptive

Number of non keratinized cells

	N	Mean	Std. Deviation	Std. Error	95% Confidence interval for mean		Minimum	Maximum
					Lower bound	Upper bound
Normal	5	3.00	2.121	0.949	0.37	5.63	0	5
Adverse oral habits	5	5.00	3.082	1.378	1.17	8.83	0	8
Diabetes	5	11.40	2.881	1.288	7.82	14.98	8	15
Total	15	6.47	4.486	1.158	3.98	8.95	0	15

 

 

Multiple comparisons

Dependent Variable: Number of no keratinized cells

Turkey HSD

* The mean difference is significant at the 0.05 level

 

Kruskal Wallis Test:

 

Table 7: Presents the micronuclei group cross tabulation of the three groups

Micronuclei* GROUP Corsstabulation

 

 

Figure 1: Presents the number of non keratinized cells of the three groups.

 

Figure 2: Presents the number of micronuclei of the three groups:

DISCUSSION:

The ratio of the nuclear to cytoplasmic area of cells in patients with adverse oral habits to that of normal, diabetes to that of normal and adverse oral habits and diabetes do not show any significant change. This indicates that there isn't a varied difference in the nuclear and the cytoplasmic volume seen in the mucosal cells of patients with these groups.

 

Based on the statistical data obtained, the P values of the three groups are significant. Kruskal wallis test gives a p value of 0.007.

 

On comparing the number of non keratinized cells observed in smears obtained from patients with diabetes to normal smears, significant variation was observed. Similar results were obtained on comparison of cells seen in normal smears to those obtained from patients with adverse oral habits.

 

On comparing the three groups, it is observed that, patients with diabetes possessed the maximum number of non keratinized cells, followed by patients with associated habits and then by patients with normal mucosa.

The percentage distribution of micronuclei indicated, absence in the mucosal cells of patients with normal mucosa. Mucosal cells of patients with diabetes, posses maximum micronuclei of about 60%. Fewer micronuclei were observed in the mucosal cells of patients with associated habits of about 40%.

       

Figure 3: Squamous cells at 10X  Figure 4: Squamous cells at 40X.

 

Figure 5: Micronuclei in a smear from diabetic patient.

 

Figure 6: Squamous cells seen in smear from a patient with associated habits stained using Papanicoloau stain.

 

CONCLUSION:

In accordance to studies conducted earlier, significant results weren't obtained in the present study. This could be due to the

reduced sample size. Hence the present study is to be continued with a larger sample size in order to obtain clear cut results, thus highlighting the dysplastic changes of the cells as well as the nuclear cytoplasmic ratio observed in

different groups.

 

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Accepted on 20.11.2016           © RJPT All right reserved