Cytological Method of Barr Body Expression in Dental Pulp Tissue at Varying Temperature

Ambika Murugesan; Sekar Balakrishnan; Indra Priyadharshini Kumaresan; Arun Jayakumar; Jayashree Mohan; Bharath Champakesan

doi:10.4103/jofs.jofs_102_18

ORIGINAL ARTICLE

Year : 2018 | Volume : 10 | Issue : 2 | Page : 108-111

Cytological Method of Barr Body Expression in Dental Pulp Tissue at Varying Temperature

Ambika Murugesan¹, Sekar Balakrishnan¹, Indra Priyadharshini Kumaresan¹, Arun Jayakumar², Jayashree Mohan³, Bharath Champakesan⁴
¹ Department of Oral Pathology, Vinayaka Mission’s Sankarachariyar Dental College, VMRF (deemed to be university), Salem, Tamil Nadu, India
² Department of Prosthodontics, Karpaga Vinayaga Institute of Dental Science, Kanchipuram, Tamil Nadu, India
³ Department of Prosthodontics, Vinayaka Mission’s Sankarachariyar Dental College, VMRF (deemed to be university), Salem, Tamil Nadu, India
⁴ Department of Community Dentistry, Vinayaka Mission’s Sankarachariyar Dental College, VMRF (deemed to be university), Salem, Tamil Nadu, India

Date of Web Publication

2-Jan-2019

Correspondence Address:
Dr. Ambika Murugesan
Department of Oral Pathology, Vinayaka Mission’s Sankarachariyar Dental college, Ariyanoor, Salem, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jofs.jofs_102_18

Abstract

Introduction: Natural disasters in large population need major attention in person identification. It can be performed by means of simple techniques that are easier to use. Aim: To identify the Barr body in female dental pulp by cytological method, after exposing at varying temperature. Objective: The expression of Barr bodies was compared among different age groups, various temperature, and two types of stain. Materials and Methods: Teeth obtained from 60 female individuals were divided into two groups based on their age. In each group, 30 teeth were exposed at three different temperatures (200°C, 300°C, and 400°C), 10 teeth in each temperature. The pulp tissue obtained from teeth was centrifuged and stained with hematoxylin and eosin (H&E) and Papanicolaou stain (PAP). Presence of visible sex chromatin in the nuclear periphery was considered positive for Barr chromatin test. The results obtained were analyzed by both descriptive analysis and independent t-test. Result: The peripherally condensed Barr body in hyperchromatic nuclear outline was clearly visible in younger individuals at 200°C and is more accurately expressed in H&E stain. Conclusion: In our study, based on the results obtained from the smear of pulp tissue, expression of Barr bodies’ decreases as age and temperature increases. Further study with increase in sample size is mandatory to confirm the above conclusion.

Keywords: Barr chromatin, H&, E stain, varying elevated temperature

How to cite this article:
Murugesan A, Balakrishnan S, Kumaresan IP, Jayakumar A, Mohan J, Champakesan B. Cytological Method of Barr Body Expression in Dental Pulp Tissue at Varying Temperature. J Orofac Sci 2018;10:108-11

How to cite this URL:
Murugesan A, Balakrishnan S, Kumaresan IP, Jayakumar A, Mohan J, Champakesan B. Cytological Method of Barr Body Expression in Dental Pulp Tissue at Varying Temperature. J Orofac Sci [serial online] 2018 [cited 2019 Jan 18];10:108-11. Available from: http://www.jofs.in/text.asp?2018/10/2/108/249083

Introduction

Forensic odontology is a branch of forensic medicine in which, the interest of justice deals with the professional handling and examination of dental evidence and with expert interpretation and documentation of the dental findings.^[1] Identification of individual is the first step in forensic investigation, and it includes diagnosis of sex, age, nutritional state, height, and other data.^[2] The teeth is durable in fire and bacterial decomposition.^[3] The pulp well protected by hard tissue serves as a valuable tool in forensic dentistry.^[4],[5],[6]

Based on the presence of Barr body, the sex of the person is indicated as female.^[7] In this study, Barr bodies were identified in extracted tooth subjected to varying temperature and were compared in two different age groups and stains.

Materials and Methods

An in vitro study was performed in pulp of extracted teeth obtained from 60 female individuals by cytological method. The individuals were divided into two age groups: group A (aged 12–30 years) and group B (aged 40–60 years). In younger individuals, teeth were obtained from orthodontic extraction of premolars and impacted molars, and in older individuals, extraction due to attrition/abrasion and mobile teeth were also included. The ethical committee of Vinayaka Mission’s Sankarachariyar Dental College, Salem (VMSDC/IEC/Approval No. 104), approved this study on February 4, 2016.

Study Design

The extracted fresh teeth were washed for removal of debris. The teeth were embedded in a metal ring and were subjected to temperatures 200°C, 300°C, and 400°C (10 teeth in each temperature for 5 min). The teeth were allowed to cool at room temperature. The pulp tissue, extirpated by sectioning the teeth, was centrifuged at 1000 rpm for 5 min. The collected pulp tissue was smeared on two sets of slides and stained by eosin hematoxylin and other set by Papanicolaou stain.

Presence of visible sex chromatin in the nuclear periphery was positive for Barr chromatin test and were categorized based on scoring criteria (score 0—absent, score 1—present but not clear, and score 2—present and clear). The statistical analysis used for comparing the expression of Barr bodies in groups A and B was performed by descriptive analysis and independent t-test.

Results

In 400°C, the tooth and the pulp were either burnt or disorganized, which did not allow further processing [Figure 1]. The results based on the scoring criteria for temperatures at 200°C and 300°C are given in [Table 1] and [Table 2].

Figure 1 Tooth is burnt and disorganized at 400°C

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Table 1 Results based on scoring criteria at 200°C

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Table 2 Results based on scoring criteria at 300°C

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At 200°C, the morphology of the tooth was not altered, and the pulp tissue shows numerous fibroblast with oval nuclei. In both the groups (A and B), the cells exhibit hyperchromatic nuclear outline with peripheral condensation of Barr chromatin [Figure 2]. In Papanicolaou stain (PAP), the expression of Barr body and the nuclear details were not clear. The mean value for hematoxylin and eosin (H&E) and PAP-stained cells in group A is 1.0 ± 0.843 and 0.80 ± 0.422, respectively, with a P = 0.001; in group B, it is 1.20 ± 1.033 and 0.60 ± 0.516, respectively, with a P = 0.005. Compared to PAP, H&E stain shows significant expression of Barr chromatin in cells of both groups A and B individuals at 200°C with a P = 0.001 [Table 3].

Figure 2 At 200°C, cytological smear of female dental pulp tissue stained with H&E showed fibroblast with condensed Barr chromatin in the periphery of the nucleus

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Table 3 Comparative analysis of cells showing clear expression of Barr chromatin in H&E and PAP stain

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At 300°C, the morphology of tooth was quite brittle, and the pulp tissue shows numerous individual fibroblast with disorganized nuclei. In group A, the cells exhibited mild hyperchromatic nuclear outline with peripheral condensation of Barr chromatin. In group B, the cells showed a disorganized nuclear outline with lack of Barr chromatin, only few cells exhibited mild peripheral condensation in nucleus [Figure 3]. The mean value for H&E and PAP-stained cells in group A is 0.73 ± 1.009 and 0.36 ± 0.505, respectively, with a P = 0.038; in group B, it is 0.80 ± 1.033 and 0.40 ± 0.516, respectively, with a P = 0.037. At 300°C when comparing the expression of Barr chromatin in PAP and H&E stain, the results were not significant (P = 0.545) in both the groups.

Figure 3 At 300°C, cytological smear of female dental pulp tissue stained with H&E stain showed fibroblast with mild peripheral condensation of Barr chromatin

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Discussion

Accidents occurring due to natural disasters lead to excessive tissue destruction in an individual and make the forensic investigation quite challenging. Tooth being the hardest tissue remains intact after death and serves an excellent tool in identification of individual, living or nonliving, for anthropological, genetic, odontologic, and forensic investigations.^[3]

Barr and Bertam’s first discovered Barr bodies, present as a mass lying against nuclear membrane measuring 1 μ in diameter.^[8] Barr chromatin is a basophilic structure with different morphological forms such as spherical, rectangular, planoconvex, biconvex, or triangular.^[9] Barr bodies are seen in 40% of female cells. Temperature and humidity affects the putrefaction of pulpal tissue.^[4] Barr bodies expression can be determined by the use of nuclear stains such as H&E, thionine, Papanicolaou, Feulgen, cresyl-violet, Giemsa, aceto-orcein, and under fluorescence such as acridine orange.^[10]

In our study, H&E and PAP stains were used because they are routinely used for cytological smears.

A number of studies were performed in Barr body identification from pulp tissue exposed to different conditions. The extracted teeth were placed in room temperature and analyzed at various scheduled time intervals in their respective studies by Dange et al.^[11] and Khorate et al.^[12] Seno^[13] and Whittaker et al.^[14] determined the Barr bodies from necrotic pulp tissue.

Expression of Barr body from dental remains is possible when the teeth are exposed at higher degree of temperature. In this study at 200°C, the cells exhibited nuclear hyperchromatism with peripheral condensation of Barr chromatin, which was similar to study conducted by Suazo et al. where the exposed pulp was histologically processed.^[15] At 300°C, the cells exhibited clear nuclear outline with low visibility of Barr chromatin. At 400°C, the pulp tissue was burnt or disorganized, which was in contradiction to the study conducted by Galdames et al.^[15] and Reddy et al.^[16]In group A, 80% of cases showed positivity for Barr chromatin at 200°C and only 40% of cases showed positivity at 300°C. Based on the above results, as the temperature increases, the cellular details decreases. Similar results were obtained by Reddy et al.^[16]

In group B, at 200°C, the Barr chromatin in cells was visualized as mild hyperchromatic condensation in nuclear periphery, and at 300°C, the number of cells with Barr body was less. Compared to PAP, H&E-stained cells showed clear Barr chromatin expression, which were similar to the results obtained by Khanna.^[2]

However, some of the limitations of our study are that the temperature and duration for which the victim exposed during an accident may not be same as the experimental in vitro temperature used in our study. In addition, temperatures above 300°C may be reached during a fire, and we found that the tooth is destroyed and not useful for investigation at such high temperatures.

Still, cytological method of Barr body identification in dental pulp tissue at varying temperatures subjected in two different age groups and stain defines the uniqueness of our study.

Conclusion

This study provides valid information that Barr chromatin identification from human dental pulp is possible when exposed at varying high temperatures and can be identified using cytological method. Advanced methods such as DNA analysis are available to determine the gender of a person in case of mass disaster, but still the cytological technique paves an easier and cost effective method in identifying the presence of Barr chromatin. However, more sample size and different conditions (such as burial, pH, frozen temperature) are required for further detailed analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Keiser-Nielsen S. Person identification by means of the teeth: A practical guide. Bristol: John Wright and Sons Ltd; 1980. pp 54-72.
2.	Khanna KS. Efficacy of sex determination from human dental pulp tissue and its reliability as a tool in forensic dentistry. J Int Oral Health 2015; 7(Suppl 2):10-6.
3.	Kaushal S, Patnaik VVG, Agnihotri G. Mandibular canines in sex determination. J Anat Soc India 2003;52:119-24.
4.	Das N, Gorea K, Gargi J, Singh JR. Sex determination from pulpal tissue. J Indian Acad Forensic Med 2004;26:50-4.
5.	Veeraraghavan G, Lingappa A, Shankara SP, Mamatha GP, Sebastian BT, Mujib A. Determination of sex from tooth pulp tissue. Libyan J Med 2010;5:1-10.
6.	Krishan K, Kanchan T, Garg AK. Dental evidence in forensic identification—An overview, methodology and present status. Open Dent J 2015;9:250-6.
7.	Sahu P, Pal SK, Rana A. Determination of age and development of Barr body in root sheath cells of human females. J Forensic Sci Crim Investig 2017;5:001-4.
8.	Mittwoch U. Sex chromatin. J Med Genet 1964;1:50-76.
9.	Anoop UR, Ramesh V, Balamurali PD, Nirima O, Premalatha B, Karthikshree VP. Role of Barr bodies obtained from oral smears in the determination of sex. Indian J Dent Res 2004;15:5-7.
10.	Sharma S, David MP, Indira AP. Gender determination from dental pulp tissue. Int J Contemp Med Sci 2017;4:821-4.
11.	Dange AH, Malvankar AG, Madiwale MS. Determination of the sex origin of teeth. Arch Kriminol 1978;162:115-9.
12.	Khorate MM, Dhupar A, Ahmed J, Dinka AD. Gender determination from pulpal tissue. J Forensic Dent Sci 2014;6:107-12. [PUBMED] [Full text]
13.	Seno M. Sex identification of human tooth by Y chromatin in the nucleus of dental pulp cell. Jpn J Legal Med 1977;31:172-9.
14.	Whittaker DK, Llewelyn DR, Jones RW. Sex determination from necrotic pulp tissue. Br Dent J 1975;139:403-5.
15.	Galdames IS, Flores A, Roa I, Cantín M, Zavando D. Sex determination by observation of Barr body in teeth subjected to high temperature. Int J Morphol 2011;29:199-203.
16.	Reddy AVS, Prakash AR, Killampalli LK, Rajinikanth M, Sreenath G, Sabiha PB. Gender determination using Barr bodies from teeth exposed to high temperatures. J Forensic Dent Sci 2017;9:44-9. [PUBMED] [Full text]