Cytomorphometric and Nucleomorphometeric analysis in Patients with Malignant Lesion, Patients with associated habits but with apparently Normal Mucosa and Control Group.

 

Harsha. L¹*, Dr. Gheena. S², Khushali. K. Shah¹

¹2nd 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 the study is to evaluate the dysplastic changes, changes in the cell and nuclear morphology and diameter in Buccal smears of patients in study groups. Background: Oral exfoliative cytology is a simple and relatively pain free procedure.Oral exfoliative cytological technique has been used for the detection of oral premalignant  malignant lesions. 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  10 patients with malignant lesion, 10 patients with associated habits but apparently normal mucosa and 10 healthy individuals (control group). All the smears were stained and evaluated using research microscope and image analysis software. Reason: To explore the potential of Exfoliative cytology as a diagnostic adjunct in patients with malignant disorders and in patients with associated habits but apparently normal mucosa.

 

 

 

 

INTRODUCTION:

Oral cancer is currently recognized as the most common malignancy in the world⁽¹⁾. The common etiological factor that accounts for these malignancies are adverse associated habits, such as smoking, alcoholism, etc⁽²⁾. This results in histological changes in the buccal mucosa which is a predisposing factor for the development of these lesions ⁽³⁾. The rapid turnover of cells play a valuable role in the diagnosis of these malignancies ⁽⁴⁾.  Early diagnosis and prompt treatment  offers best hope in improving the prognosis in patients^(5).

 

Exfoliative cytology is a simple, non invasive diagnostic technique, that increases the chance of earlier detection of dysplastic changes in the cells⁽⁶⁾. With advancements in the field of quantitative oral exfoliative cytology, this technique has gained importance in the diagnosis of malignancies.

 

Parameters such as nuclear size, cell size, nuclear cytoplasmic ratio, presence of micronuclei, nuclear shape, nuclear discontinuity, optical density and nuclear texture can be evaluated to establish the appropriate diagnosis of malignancy⁽⁷⁾ The present study aims at analyzing the cytomorphometric  and nucleomorphometric changes in the cells of the buccal mucosa obtained via exfoliative cytological technique in patients with malignant lesions and in patients with associated habits.

 

MATERIALS AND METHODS:

The study group consisted of 15 patients, 5 patients with  malignant disorders, 5 patients with associated habits but with apparently normal mucosa and 5 control group.

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

 

Patients with malignant lesion:

Inclusion criteria:

Patients aged 45 years and above.

Patients with a  malignant lesion in the oral cavity.

Patients undergoing radiation and chemotheraphy.

Patients diagnosed with cancer.

Patients with associated habits but apparently normal mucosa:

Inclusion criteria:

Patients aged 45 years and above

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

Patients who have regular drinking habits for the past 10 years, with apparently normal mucosa.

 

Exclusion criteria:

Patients who suffered from systemic diseases like anemia, diabetes, etc.

Patients who have undergone radiation therapy or chemo therapy.

Patients with malignant or  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 do not have any systemic complications.

Patients who did not have oral lesions.

Patients with poor oral hygiene were excluded.

 

Method for collection of data:

Smears were collected from patients of the specific group using a wooden spatula moistened in distilled water. Three smears were obtained from the buccal mucosa of each patient included in the study. The scrapings were transferred onto a clean glass slide, to produce a thin and uniform smear. The prepared slides were fixed in 95% propanol.

 

Staining: two slides were stained using hematoxylin and eosin and the other one with papanicolaou stain.

 

The stained slides were then visualized under the microscope to study the cytomorphometrics and nucleomorphometrics of the cells- cytoplasmic area, nuclear area and nuclear cytoplasmic ratio.

 

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

 

RESULTS:

The present study includes 5 samples obtained from each group.

 

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
MALIGNANT LESION
S.NO	NUCLEAR AREA(µm2)	CELL AREA (µm2)
T1	102	2098
T2	98	1890
T3	134	3046
T4	146	4126
T5	134	3198
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

 

 

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.

MALIGNANCT LESION	Number of keratinized cells	Number of non keratinized cells	micronuclei	Number of cells undergoing keratinisation
T1	0	0	+++	20
T2	0	8	++	12
T3	10	0	+	10
T4	0	5	+	15
T5	3	3	++	14
ADVERSE ORAL HABITS	Number of keratinized cells	Number of non keratinized cells	micronuclei	Number of cells undergoing keratinisation
A1	0	0	+++	20
A2	4	7	+++	9
A3	0	8	+	12
A4	0	5	+	15
A5	0	5	++	15
NORMAL	Number of keratinized cells	Number of non keratinized cells	micronuclei	Number of cells undergoing keratinisation
N1	0	0	Nil	20
N2	0	5	Nil	15
N3	2	3	Nil	15
N4	0	5	Nil	15
N5	0	2	+	18

Table 3: Presents the comparison of the cellular and nuclear area of normal to malignant lesions

	SAMPLE	N	Mean	Std. Deviation	Std. Error Mean	‘t’	Sig
Nuclear area	Normal	5	123.2000	18.37662	8.21827	.032	.976
	PMOD	5	122.8000	21.42895	9.58332
Cell area	Normal	5	3496.6000	548.92377	245.48617	1.321	.223
	PMOD	5	2871.6000	904.49809	404.50384
RATIO	Normal	5	28.3729	.87714	.39227	3.392	.009
	PMOD	5	22.9423	3.47070	1.55214

 

Table 4: Presents the comparison of the cellular and nuclear area of normal to adverse oral habits.

	SAMPLE	N	Mean	Std. Deviation	Std. Error Mean	‘t’	Sig
Nuclear area	Normal	5	123.2000	18.37662	8.21827	1.799	.110
	Adverse oral habits	5	138.4000	4.39318	1.96469
Cell area	Normal	5	3496.6000	548.92377	245.48617	.591	.571
	Adverse oral habits	5	3708.8000	586.72327	262.39062
RATIO	Normal	5	28.3729	.87714	.39227	.994	.349
	Adverse oral habits	5	26.7284	3.59311	1.60689

 

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

	SAMPLE	N	Mean	Std. Deviation	Std. Error Mean	‘t’	Sig
Nuclear area	Adverse oral habits	5	138.4000	4.39318	1.96469	.822	.149
	PMOD	5	122.8000	21.42895	9.58332
Cell area	Adverse oral habits	5	3708.8000	586.72327	262.39062	.819	.121
	PMOD	5	2871.6000	904.49809	404.50384
RATIO	Adverse oral habits	5	26.7284	3.59311	1.60689	.393	.129
	PMOD	5	22.9423	3.47070	1.55214

 

 

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

 

Kruskal-Wallis Test

 

 

 

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

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

 

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

 

Comparison of the nuclear to the cytoplasmic area of the cells obtained from the three groups was done using T test. Comparison of the number of non keratinized cells among the three groups was done using Tukey HST and Kruskal-Wallis Test. Micronuclei cross tabulation was done to obtain the percentage distribution of micronuclei among the three groups.

 

DISCUSSION:

The ratio of the nuclear to cellular area of cells, in patients with malignant lesions shows a significant change to that of normal. This indicates that there a varying changes in the nuclear and the cytolplasmic volume of the mucosal cells present in patients with malignant oral lesions. In a study conducted by Shabana et al,  a steady increase in  the nuclear and the cytoplasmic area was indicated, which is  in accordance to the present study⁽⁸⁾.

 

The ratio of the nuclear to cytoplasmic area of cells in patients with adverse oral  habits to that of normal 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 adverse oral habits.

 

On comparing the ratio of the nuclear to cellular volume of cells in patients with malignant lesion and adverse oral habits, there is no significant variation observed.

 

Based on the statistical data obtained, the P value of the three groups are not significant. Kruskal wallis test gives a p value of 0.447, which is not significant. Though the number of non keratinized cells vary, statistical variation was not obtained.

 

On comparing the number of non keratinized cells observed in smears obtained from patients with malignant lesion to normal smears, no 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 adverse oral habits possessed the maximum number of non keratinized cells, followed by patients with malignant lesions 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 adverse oral habits, posses maximum micronuclei of about 40%. Fewer micronuclei were observed in the mucosal cells of patients with malignant lesions.

 

CONCLUSION:

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

 

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Accepted on 27.07.2016           © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(10):1699-1703.