Analysis of erythrocytes in oral submucous fibrosis – A morphometric study

Vijay Wadhwan; Arvind Venkatesh; Preeti Sharma; Sangeeta Malik; Chitrapriya Saxena

doi:10.4103/ijdr.IJDR_718_18

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ORIGINAL RESEARCH

Year : 2021 | Volume : 32 | Issue : 1 | Page : 27-30

Analysis of erythrocytes in oral submucous fibrosis – A morphometric study

Vijay Wadhwan¹, Arvind Venkatesh², Preeti Sharma¹, Sangeeta Malik³, Chitrapriya Saxena⁴
¹ Department of Oral Pathology & Microbiology, Subharti Dental College, Meerut, Uttar Pradesh, India
² Department of Oral and Maxillofacial Pathology, Smile Square Multispecialty Dental Centre, Karur, Tamil Nadu, India
³ Department of Oral Medicine and Radiology, Subharti Dental College, Meerut, Uttar Pradesh, India
⁴ Department of Oral Pathology & Microbiology, LLRM Medical College, Meerut, Uttar Pradesh, India

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Date of Submission	07-Oct-2018
Date of Decision	11-Jan-2020
Date of Acceptance	30-Oct-2020
Date of Web Publication	13-Jul-2021

Abstract

Background: Oral submucous fibrosis (OSMF) is a chronic progressive scarring oral disease caused due to areca quid chewing. The constituents of areca nut can enter into the circulation by swallowing the liquid mixture of areca quid which evokes oxidative stress on RBC membrane integrity. Aims: To study the morphometric characteristics of erythrocytes under light microscopy and to assess the role of areca quid constituents on the morphology of circulating erythrocytes. Materials and Methods: Peripheral blood smears prepared from blood samples of 50 patients selected according to Lai's classification. Group I: Normal individuals (10 cases); Group II: Mouth opening > 35 mm (10 cases); Group III: Mouth opening 30-35 mm (10 cases); Group IV: Mouth opening 20-30 mm (10 cases) and Group V: Mouth opening < 20 mm (10 cases). The slides were stained with Leishman's stain and assessed by light microscopy. A total of 100 randomly selected RBCs from 5 different fields in each smear were selected and the RBC circumference was measured and tabulated. Statistical analysis and Results: Data was analyzed using GraphPad Prism 5.03 software. Tukey's multiple comparison test showed statistically significant difference (p < 0.05) between groups I and IV; I and V; II and IV; II and V; III and IV; III and V. Conclusion: These results suggest the possibility of cytotoxic effect of areca quid constituents on circulating erythrocytes in advanced cases of OSMF, which might result in microcytic anaemia.

Keywords: Erythrocytes, peripheral blood smear, submucous fibrosis

How to cite this article:
Wadhwan V, Venkatesh A, Sharma P, Malik S, Saxena C. Analysis of erythrocytes in oral submucous fibrosis – A morphometric study. Indian J Dent Res 2021;32:27-30

How to cite this URL:
Wadhwan V, Venkatesh A, Sharma P, Malik S, Saxena C. Analysis of erythrocytes in oral submucous fibrosis – A morphometric study. Indian J Dent Res [serial online] 2021 [cited 2023 Mar 26];32:27-30. Available from: https://www.ijdr.in/text.asp?2021/32/1/27/321383

Introduction

Oral submucous fibrosis (OSMF) is a chronic progressive scarring oral disease predominantly affecting people of South Asian origin. It is characterized by juxtaepithelial inflammatory cell infiltration followed by fibrosis in the lamina propria and submucosa of the oral mucosa. Epidemiologic studies have suggested the habit of areca quid chewing as a major etiologic factor of OSMF.^[1] Most convincing evidence is derived from case-control studies that estimate the odds ratios for areca nut use among oral submucous fibrosis cases and a definite dose-dependent relationship between areca nut and causation of the disease. Daily use appeared to be more important than the duration of the habit.^[2] The copper content of areca nut is high and the levels of soluble copper may rise in volunteers who chew areca quid (302 nmol/g).^[3] The possible role of copper as a mediator of fibrosis is supported by the demonstration of upregulation of this enzyme in OSMF biopsies.^[4] Animal experiments conducted to assess the metric parameters of erythrocytes in blood after influence of different copper concentrations showed the beginning of processes of atrophy in the low copper concentrations and necrosis in the higher copper.^[5] Experiment conducted to demonstrate the cytotoxic effects exerted by the mixed constituents' present in areca nut suggested the constituents of areca nut can enter into the circulation by swallowing the liquid mixture of betel nut chewing, which evokes oxidative stress on erythrocyte membrane integrity accompanied by drastic morphological changes in the Areca Nut extract (ANE) treated erythrocytes at ultrastructural level,^[6] but the effect of areca quid constituents on the circulating erythrocytes at light microscopic level has been seldom analysed. Hence, this study was designed to study the morphometric characteristics of erythrocytes under light microscopy and to assess the role of areca quid constituents on the morphology of circulating erythrocytes.

Subjects and Methods

Selection of cases

The material for the study consisted of blood samples collected from 36 patients clinically diagnosed with oral submucous fibrosis. Blood samples of 10 normal patients were obtained and used as control. An informed consent was obtained from all the participants of the study. Ethical approval to conduct the study was obtained from the Institutional Ethical Committee. The samples obtained were further divided into various groups based on the inter-incisal distance which was obtained during clinical examination and subdivided as Group I: Normal individuals (Control) (10 cases) without any history of chewing betel nut; Group II: Mouth opening >35 mm (10 cases); Group III: Mouth opening 30-35 mm (10 cases); Group IV: Mouth opening 20-30 mm (10 cases) and Group V: Mouth opening <20 mm (06 cases) according to Lai et al. classification of oral submucous fibrosis.^[7] Only male patients had been included for the study to overcome gender bias. The age of the patients included for the study were between 17 and 48 years.

Sample collection and preparation of the blood smear

Blood samples of patients falling under any of the various case groups of the study population collected for routine blood examination before dental and minor surgical procedures were only used for the study after obtaining informed consent from the patient. 2 ml of blood sample was collected from the antecubital vein, and then transferred to specimen containers containing K3 Ethylene Diamine Tetra Acetic acid (K3 EDTA).

Smear preparation and staining

Blood smears were made by manual procedure on the glass slides. The peripheral blood smears were stained using Leishman's stain by the following procedure. The peripheral blood smears prepared were air dried and flooded with stain. After 2 minutes, double the volume of buffered water (pH – 7.2) was added to the slide for 10 minutes. Slides were washed in a stream of buffered water until it acquired a pinkish tinge.^[8]

Slides were placed to dry and then were subjected to light microscopic examination.

Morphometric analysis

Each slide was assessed by light microscopy. 5 suitable areas of the blood film in which there was no rouleaux formation and the red cells that were touching but with little overlap were selected and images were captured under x400 magnification. 100 randomly selected cells from 5 different fields (20 cells/field) in each blood smear was measured in a stepwise pattern in which the erythrocyte circumference was obtained by drawing around the cell boundaries using the digitaliser cursor and the measuring mode of Image Pro Express software version 6.0 [Figure 1]. The circumference of erythrocytes was measured from all the fields of each peripheral blood smear prepared and the mean erythrocyte circumference in microns of each patient were tabulated [Table 1].

Figure 1: Morphometric analysis of RBC as on Peripheral Blood smear

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Table 1: Mean erythrocyte circumference in various groups

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Statistical analysis

The mean erythrocyte circumference of different groups were analysed using One- way ANOVA and Tukey's multiple comparison test. P values of <0.05 were considered to be statistically significant.

Results

Data were analysed using GraphPad Prism software version 5.03 for Windows (GraphPad Software, Inc. La Jolla, California).

The mean erythrocyte circumference of Group I was 30.573 μ ± 0.37559 μ (Mean ± Standard Deviation); Group II was 30.602μ ± 0.23517 μ; Group III was 30.212 μ ± 0.49263 μ; Group IV was 29.443 μ ± 0.85418 μ and Group V was 29.383 μ ± 0.6653 μ [Table 1].

A statistically significant difference was noted in the mean erythrocyte circumference between Groups I, II, III, IV and V (One way ANOVA; P< 0.0001). Furthermore, statistical significance at P < 0.05 was noted between Groups I and IV; I and V; II and IV; II and V; III and IV; III and V respectively using Tukey's multiple comparison test [Table 2].

Table 2: Tukey's multiple comparison test

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Discussion

OSMF is a chronic disorder characterized by fibrosis of the lining mucosa of the upper digestive tract involving the oral cavity, oropharynx and frequently the upper third of the esophagus. OSMF is well recognized as a potentially malignant disorder.^[9] There is compelling evidence to implicate the habitual chewing of areca nut with the development of OSMF.^[10] The seed contains alkaloids such as arecaidine and arecoline, and in addition consists of carcinogens like tannins.^[11] The copper content of areca nut is high (302 nmol/g) and the levels of soluble copper in saliva may rise in individuals who chew areca quid.^[3],[12]

Erythrocytes are highly differentiated cells that have no nuclei or cytoplasmic organelles. Normal erythrocytes are circular biconcave discs with a mean diameter of 7.2 μm (range 6.7–7.7 μm) in dried fixed smears and about 7.5 μm in the living state. They are eosinophilic and consequently appear red with a central area of pallor in Romanowsky-stained smears.^[13]

The membrane of the red blood cell plays many roles that aid in regulating their surface deformability, flexibility, adhesion to other cells and immune recognition. These functions are highly dependent on its composition, which defines its properties. A Red Blood Corpuscle (RBC) membrane has two main components: a lipid bi-layer (similar to those found in most human cells) and a protein skeleton. The skeleton forms an extensive web around the bilayer and is joined to it at what are called junctional complexes.^[14]

Studies suggest that there are specific sulfhydryl groups that are intimately related to membrane structure and are relatively sensitive to in vivo and in vitro oxidation, or to in vitro blockade by agents that react with sulfhydryl groups. The maintenance of these groups in the reduced state is dependent on metabolic processes within the erythrocyte. It has been postulated, furthermore, that the critical changes affecting erythrocyte life span occur in the sulfhydryl groups of the erythrocyte membrane.^[15]

An in vitro study conducted by Peng et al. to evaluate the effect of areca nut extract on isolated RBC's showed the various constituents of areca nut extract can enter into the circulation by swallowing the liquid mixture of betel nut chewing, an oxidative-stress-evoked effect on RBC membrane integrity can ensue.^[6]

This oxidative stress created by areca nut extract constituents might result in inhibition of the sulfhydryl groups of the RBC membrane by covalent bonding of the constituents like arecoline and heavy metals to sulfhydryl groups on the membrane^[16] which would result in the loss of sulfhydryl activity. Peng et al. also noticed that areca nut extract caused a concentration-dependent reduction in membrane sulfhydryl groups.^[6] It is suggested that a loss of membrane sulfhydryl activity may be the decisive haemolytic event.^[17]

There are two pathways that could help to interpret the possible mechanism(s) associated with the ANE-induced RBC membrane sulfhydryl depletion. First, the depletion of membrane sulfhydryl groups can proceed via ANE provoked reactive oxygen species production that is preceded by the oxidation reaction of membrane thiols. Alternatively, the constituents in ANE such as arecoline and eugenol could also form conjugates with the membrane thiol groups. The decrease in membrane deformability by ANE can also be explained by the effect of ANE on membrane thiols. It has been documented that alkylation of sulfhydryl groups by N-ethylmaleimide can alter spectrin self-association, which leads to instability of the membrane skeleton.^[18]

An in vitro conducted by Kohle et al. to study the morphological aberration and cytotoxicity of areca nut extract on RBC's showed hemolysis with 5 ug/ml concentration or above. A concentration dependent increase in haemolysis was noted between 5 ug/ml and 100 ug/ml .The maximum haemolysis occurred at 100 ug/ml concentration of aqueous extract of areca nut.^[19] Peng et al. also revealed drastic morphological changes of ANE-treated RBCs in a dose and time dependant manner.^[6]

Based on these data from literature, we sought to study the cytotoxic effects of areca nut extract on circulating erythrocytes morphometrically under light microscopy. The present study revealed significant difference in the size (circumference) of RBC between normal individuals (Group I), mild cases of OSMF (Group II) and severe cases of OSMF (Groups IV and V).

This might be indicative of the alteration in the membrane composition in the circulating RBC's which in turn is suggestive of cytotoxic effects induced by ANE on erythrocytes. But the exact pathogenesis involved in the role of areca nut extract on circulating RBC's is seldom studied.

Microcytic hypochromic anaemia has been reported in patients with OSMF^{[20],[21],[22]} but there has been no definite proof proposed to support this cause.^[23]

The results of the present study indicate that there is a constant decrease in the mean circumference of the erythrocyte across the groups from milder forms to more severe forms of OSMF [Table 1]. This indicates that there will be corresponding reduction in the Mean Corpuscular Volume (MCV) of the erythrocyte which would ultimately lead to decrease in the Mean Corpuscular Hemoglobin Concentration (MCHC). As oxidative stress induced by arecoline from the ANE is attributed to the loss of erythrocyte membrane integrity,^[6] this can also be an underlying cause of the occurrence of microcytic anaemia in patients with OSMF.

Conclusion

Based on the results of this study we propose that similar studies need to be conducted with larger sample sizes to validate the results obtained. Further, studies at electron microscopic level have to be conducted to evaluate the ultrastructural changes in the erythrocytes; molecular studies to evaluate the underlying pathophysiology have to be carried on. Longitudinal studies have to be performed in order to evaluate long term morphological and morphometric changes and to study if these changes reverse following the cessation of habit or treatment. This might probably serve as a tool to determine the progression of the disease and to evaluate the systemic effects caused by oral submucous fibrosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Dr. Vijay Wadhwan
Professor & Head, Department of Oral & Maxillofacial Pathology & Oral Microbiology, Subharti Dental College, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh
India