JIOH on LinkedIn JIOH on Facebook
  • Users Online: 179
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL RESEARCH
Year : 2020  |  Volume : 12  |  Issue : 2  |  Page : 145-152

Effect of exposure to variable degrees and durations of heat on dental Barr body identification in females: An in vitro cross-sectional study


1 Department of Forensic Medicine and Clinical Toxicology, Tanta Faculty of Medicine, Tanta University, Tanta, Gharbia, Egypt
2 Oral Biology Department, Faculty of Dentistry, Tanta University, Tanta, Gharbia, Egypt

Date of Submission12-Sep-2019
Date of Acceptance02-Nov-2019
Date of Web Publication28-Mar-2020

Correspondence Address:
Dr. Sarah Yasser
Oral Biology Department, Faculty of Dentistry, Tanta University, Tanta, Gharbia.
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_239_19

Rights and Permissions
  Abstract 

Aim: In developing countries DNA analysis techniques might be a slightly expensive forensic tool so the use of Barr body for gender determination could be a more economic method. The aim of this study is to evaluate the effect of exposure to variable degrees and durations of heat on dental pulp Barr bodies identification in human females. Materials and Methods: 130 premolars were divided equally into control and 12 study groups. The study groups were heated to different temperature degrees (100° c, 200° c and 300° c) for duration intervals (5, 15, 30 and 60 minutes), then the Barr body was examined in the pulp tissues using light microscope. Results: There was a significant decrease in the percentage of positive Barr body cells at 100°c, except in 5 and 15 minutes, and at 200° c groups except in 5min. Interestingly, at 200°c, Barr bodies could be identified only at 5 minutes duration. At 300°C, the pulp tissue could not be retrieved. Also, there was a significant negative correlation between the degree of heat exposure and the percentage of positive Barr body cells in the dental pulp (r = −0.73, P = < 0.001) and between the duration of exposure at each degree (100° c and 200° c) and the percentage of positive Barr body cells (r = −0.90, P = < 0.001) in 100 °c groups and in 200°c groups (r = −0.76, P = <0.001). Conclusion: Dental Barr bodies can be useful for gender determination up to 200°C at the time period of 5 min in vitro.

Keywords: Barr Body, Dental Pulp, Forensic Odontology, Heat Exposure


How to cite this article:
El-Sarnagawy GN, Yasser S. Effect of exposure to variable degrees and durations of heat on dental Barr body identification in females: An in vitro cross-sectional study. J Int Oral Health 2020;12:145-52

How to cite this URL:
El-Sarnagawy GN, Yasser S. Effect of exposure to variable degrees and durations of heat on dental Barr body identification in females: An in vitro cross-sectional study. J Int Oral Health [serial online] 2020 [cited 2020 May 28];12:145-52. Available from: http://www.jioh.org/text.asp?2020/12/2/145/281491


  Introduction Top


Fire accident deaths might occur in variant circumstances as in transportation injuries, terrorist attacks, and in electric accidents. In these situations, personal identification is difficult where victims are charred and have extensive degree of tissue damage.[1] Personal identification is a crucial process for legal and humanitarian resolutions. In addition, gender determination is a vital step in establishment of person’s individuality.[2]

Teeth remain stable even after exposure to fire disasters because they are the most calcified part in the body. Therefore, teeth are considered an excellent tool for personal identification in forensic investigations.[3]

Forensic odontology is a branch of forensic medicine, which use dental tissues for personal identification.[4] It has a significant role in identification of highly mutilated and dismembered bodies as in fire accidents, bomb explosions, and aircraft accidents.[5]

In forensic dentistry, gender determination can be performed by a variety of methods such as teeth morphology, estimation of crown diameter, and root length. In addition, microscopic methods include determination of intranuclear structure, Barr body, as Barr and Bertam[6] first discovered it. This Barr body presents as a mass condensation under the feminine nuclear membrane. About 40% of female cells contain Barr bodies, whereas male cells are considered chromatin negative.[7],[8]

Dental pulps compromise the innermost soft-tissue core of teeth that are protected by mineralized dental tissues. Consequently, pulp-tissue histological examination of Barr body is suitable in certain circumstances such as natural disasters and fire deaths where other soft tissues cannot be investigated.[9]

Accordingly, this study aimed to evaluate the effect of exposure of variable degrees and durations of heat on dental pulp Barr body identification in female as a tool for gender identification.


  Materials and Methods Top


Study design

An in vitro cross-sectional study was carried out in the Department of Oral Histology, Faculty of Density, Tanta University from July 2018 to December 2018. Sample size was calculated by G Power (version 3.1.9.4, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) depending on the primary outcome. The required sample size per group was 10 according to these assumptions; effect size of 0.4.5% margin of error, power of 80%, and number of the studied groups was 12. The procedures were in accordance with the ethical standards of the Research Ethics Committee of Faculty of Medicine, Tanta University and with the Helsinki Declaration. The approval code number was 330031/03/18. Written informed consent was obtained from each subject or her guardians. Confidentiality of the data was maintained by using coding number for each subject.

Materials

One hundred and thirty premolars of healthy female volunteers aged between 12 and 20 years were included in this study. The normal, healthy teeth were extracted during orthodontic purposes. Subjects with sexual disorders such as Klinefelter or Turner syndromes were excluded from this study. Moreover, teeth with any dental pathology, developmental abnormalities, and teeth with severe trauma or fractures were not considered also.

Sample preparation

The collected teeth were cleaned immediately after extraction with sterile distilled water. They were divided equally into control group and 12 study groups (10 teeth per each group). The study groups were heated to different temperature degrees (100°C, 200°C, and 300°C) for various duration intervals (5, 15, 30, and 60min). They were placed at room temperature to cool down; the pulp tissues were extracted by longitudinal sectioning of the teeth using a rotating disc with a micro motor spray. Fixation of pulp tissue was performed in 10% formalin. Then it was paraffin embedded, sectioned, and stained with hematoxylin and eosin stains. The sections were examined under light microscope (Leica, Wetzlar, Germany) at magnification of ×1000. The analysis procedure was carried out by counting 50 cells per slide according to Suazo et al.[10] The percentages of Barr body positive cells were counted among the total cells. The cells were considered Barr body positive by presence of chromatin condensation under nuclear membrane.[7]

Statistical analysis

Statistical analysis and presentation of data were conducted using IBM SPSS Statistics for Windows, version 22 (IBM Corp., Armonk, NY, USA), version 22.0. For quantitative data, the Kruskal–Wallis test was used for comparing between groups. In addition, Spearman’s correlation was analyzed between percentages of Barr body positive cells and various degrees and time intervals in the studied groups. Significance was adopted at P < 0.05 for interpretation of the tests results.[11]


  Results Top


One hundred and thirty premolars teeth of healthy female volunteers were included in this study. Their age ranged from 12 to 20 years with a mean of 16.37 ± 2.18 years.

Macroscopic examination of the teeth

At 100°C for 5-, 15-, and 30-min groups, the external morphological appearance of the teeth did not alter much. However, at 100°C for 60-min group and all 200°C groups, the teeth were more brittle. Furthermore, at 300°C groups the teeth showed brownish discoloration and were extremely brittle and the pulp tissue could not be retrieved.

Histological examination of dental pulp tissues

Histological examination of the control group revealed normal female dental pulp cells with the characteristic condensation of sexual chromatin (Barr bodies) [Figure 1]. At 100°C for 5 and 15min, the pulp tissue was intact and the dental pulp cells showed basophilic hyper chromatic nuclei with Barr bodies appearing as peripheral chromatin condensation with surrounding disorganized collagen fibers [Figure 2]A and B. At 100°C for 30min, the pulp tissue showed hypocellularity with a sparsely arranged collagenous stroma and the nuclei continued to have identifiable Barr bodies at their periphery though at much lower percentages than the lower durations [Figure 3]A. At 100°C for 60min, the extracted pulp-tissue volume was significantly reduced and the consistency of the tissue was altered as it had a crumbled appearance so centrifugation of the tissue for histological preparation was needed. Histologically, the pulp tissues showed features such as hypocellularity, disorganized collagenous stroma, and signs of extracellular matrix hyalinization. However, the identification of Barr bodies in the pulp cells was still possible [Figure 3]B.
Figure 1: Light micrograph showing control female dental pulp cells with the characteristic condensation of sexual chromatin. Barr bodies (black arrows). (H&E stain original magnification ×1000)

Click here to view
,
Figure 2: Light micrograph showing female dental pulp cells at (A) 100°C for 5-min group and (B) 15-min group with the characteristic condensation of sexual chromatin. Barr bodies (black arrows). (H&E stain, original magnification ×1000)

Click here to view
,
Figure 3: Light micrograph showing female dental pulp cells at (A) 100°C for 30-min group with Barr bodies (black arrows) and (B) 60-min group with the characteristic condensation of sexual chromatin. Barr bodies (black arrows) and hyalinization of the extracellular matrix (black star). (H&E stain, original magnification ×1000)

Click here to view


At 200°C groups, the amount of the extracted pulp tissue was reduced remarkably in comparison with the 100°C groups. Histologically, at 200°C for 5-min group there was obvious signs of extracellular matrix hyalinization and cells nuclei assumed a more granular appearance; however, some of the nuclei showed signs of degeneration. Interestingly, the nuclei still had identifiable Barr bodies at their periphery [Figure 4]B. At 200°C for 15-min group, the extracellular matrix showed signs of extreme hyalinization and denaturation. Also, the cell nuclei showed signs of degeneration, decomposition, and the Barr bodies could not be detected [Figure 4]B. At 200°C for 30- and 60-min groups, there was complete tissue disarray and no cells were detected, so it was not possible to establish the presence of sex chromatin [Figure 5]A and B.
Figure 4: Light micrograph showing female dental pulp cells at (A) 200°C for 5-min group with Barr bodies (black arrows) and hyalinization of the extracellular matrix (black stars) and (B) 15-min group where Barr bodies could not be identified in the nuclei and extensive hyalinization of the extracellular matrix was detected (black stars). (H&E stain, original magnification ×1000)

Click here to view
,
Figure 5: Light micrograph showing female dental pulp cells at (A) 200°C for 30-min and (B) 60-min group. There was complete tissue disarray and no cells were detected, so it was not possible to establish the presence of sex chromatin. (H&E stain, original magnification ×1000)

Click here to view


In all groups of 300°C, histological examination could not be performed as the pulp tissue could not be retrieved.

Statistical analysis of positive Barr body cell percentage in the studied groups

The median percentage of positive Barr body cells in control group was 60%. In 100°C groups, the median percentages of positive Barr body cells were 51.85%, 12.73%, 7.89%, and 7.46% in the duration 5, 15, 30, and 60min, respectively. Regarding 100°C groups, the percentages of positive Barr body cells in both of 30- and 60-min groups were significantly decreased than control group (P = 0.005 and 0.002, respectively). In addition, the 60-min group had significantly lower percentage of positive Barr body cells as compared to 5-min group (P = 0.030) [Table 1]. In 200°C groups, the median percentage of positive Barr body cells was 14.28% in 5-min group. However, no positive Barr body cells were observed in other groups. There was a significant decrease in the percentage of positive Barr body cells in the 15-, 30-, and 60-min groups as compared to control group (P = 0.003) [Table 2]. Therefore, there was a significant decrease in the percentage of positive Barr body cells in 100°C, except in the 5- and 15- min group, and in 200°C groups as compared to control group, except in 5 minutes group.
Table 1: Kruskal–Wallis test for comparison between variable duration of heat exposure at 100°C on percentage of positive Barr body cells

Click here to view
,
Table 2: Kruskal–Wallis test for comparison between variable duration of heat exposure at 200°C on percentage of positive Barr body cells

Click here to view


There was a significant negative correlation between the degree of heat exposure (100°C, 200°C, and 300°C) and the percentage of positive Barr body cells in the dental pulp (r = −0.73, P = < 0.001) [Figure 6]. Furthermore, there was a significant negative correlation between duration of exposure at each degree (100°C and 200°C) and the percentage of positive Barr body cells in 100°C groups (r = −0.90, P = < 0 .001) and in 200°C groups (r = −0.76, P = < 0 .001) [Figure 7].
Figure 6: Spearman’s correlation between percentage of positive Barr body cells and the degree of heat exposure in 100°C, 200°C, and 300°C

Click here to view
,
Figure 7: Spearman’s correlation between percentage of positive Barr body cells and duration of heat exposure in each of 100°C (A) and 200°C (B)

Click here to view



  Discussion Top


Fire accidents, terror attacks, transportation accidents, and sometimes murder by arson can leave charred human remains beyond recognition.[12] In such cases, forensic identification is a hard task because of the extensive destruction of tissues. However, the victims’ teeth are usually preserved making them an available tool for forensic identification. For gender determination, teeth can be investigated by their external morphology, polymerase chain reaction-DNA analysis and pulp-tissue examination of Barr body. Identification of Barr body in the dental pulp is considered a reliable guide for gender determination when the teeth have suffered harsh physical conditions, such as incineration and dumping.[13]

There are many advantages of using dental pulp tissues in such conditions as the teeth are encased in alveolar bone that is surrounded by gingival and other soft tissues. Moreover, the soft dental pulp is surrounded by hard dental tissues including enamel, the hardest tissue in the human body, which preserves it from the heat effect.[14] Furthermore, the use of dental pulp features is an easy, quick, and economic way than other expensive techniques.[1]

This study showed almost normal morphological appearance of the teeth subjected to 100°C for 5, 15, and 30min. On the other hand, at 100°C for 60 minutes and 200°C groups the teeth were more brittle. Moreover, at 300°C groups the teeth showed brownish discoloration and were extremely brittle. These results coincide with that of Amin et al.,[15] who showed that when teeth were heated, for 20min, at 100°C they did not show any changes in the consistency of enamel, dentin, or cementum. On the contrary, they found loss of translucency in most of the tooth samples at 200°C. Moreover, at 500°C, the teeth were carbonized and charred. Meanwhile, Reddy et al.[1] reported that the morphology of the teeth did not alter in 100°C and 200°C for 5min, although the teeth were brittle when heated at 400°C for 5min.

Also, we found that the extirpated pulp tissue was almost intact at 100°C for 5 and 15min. However, it showed reduction in volume and hypocellularity in 100°C for 30 and 60min. Meanwhile, the amount of the extirpated pulp tissue was reduced significantly in 200°C groups with signs of extracellular matrix hyalinization and could not be retrieved in 300°C groups. These findings are in line with those of Amin et al.[15] and Reddy et al.,[1] who found that the pulp tissue was intact at 100°C for 5 and 20min. However, it showed multiple degeneration and hypoceluraity at 200°C and hyalinization at 400°C and 500°C.

The median percentage of positive Barr body cells in control group was 60% in this study. This finding is in agreement with that of Khanna[16] who found 51.4% of positive Barr cells in normal dental pulp tissue. However, previous studies by Yunis and Chandler,[17] Das et al.,[8] and Suazo et al.[10] reported less frequent percentage of positive Barr body in normal dental pulp (30%, 24.92%, and 20%, respectively). These variations in results could be attributed to the adherence of Barr body to nuclear membrane that may be hidden in front or behind nucleoplasm; thus, Barr body may be not be easily identified.[10]

Furthermore, this study showed that at 100°C, the median percentages of identified Barr bodies were 51.8%, 12.7%, 7.9%, and 7.5% in the duration 5, 15, 30, and 60min, respectively. These findings are consistent with previous findings by Duffy et al.,[13] who showed preserved Barr bodies in heated pulp tissues up to 100°C for 60min and in dehydrated pulp tissues up to one year.

Although the pulp tissue could be retrieved in this study at all experimental durations at 200°C, Barr bodies could be identified only at the 5-min duration with a median percentage of 14.3%. Interestingly, at 200°C for 15-min group, the pulp cell nuclei showed signs of degeneration and decomposition and the Barr bodies could not be identified. At 200°C for 30- and 60-min groups, no nuclei were detected. Consequently, it was not possible to establish the presence of sex chromatin. Interestingly, at 300°C groups the teeth were not suitable for further pulp-tissue extraction.

These results are not in line with those of Suazo et al.[10] and Reddy et al.,[1] who reported that Barr bodies could be identified in the dental pulp up to 400°C. This could be explained by the difference in the methodology as in both studies the teeth were held in a metal crucible in which a proportion of heat energy is lost in the crucible base and walls.[18] On the contrary, in this study the teeth were put in open perforated metal trays directly in the furnace that can make the direct effect of heat to be more exaggerated.

Reddy et al.[1] found that the average percentage of Barr body positive cells remained constant (40%) with an increase in temperature at 100°C, 200°C, and 400°C. In contrast with these findings, we found that there was a significant negative correlation between the degree of heat exposure (100°C, 200°C, and 300°C) and the percentage of positive Barr body cells in the dental pulp (r = −0.73, P = < 0.001). Likewise, Suazo et al.[10] recorded a decrease in the percentage of positive Barr body cells from 30% at 200°C to 22% at 400°C. Also, Das et al.[8] showed that positive Barr body cells were ideally identified at 36.5°C and that the number of these cells decreased with further increase in temperature.

In addition, this study showed a significant negative correlation between duration of heat exposure at each degree and the percentage of positive Barr body cells, which is considered a new finding. This decrease of the percentage of positive Barr body cells could be attributed to decrease in pulp-tissue volume and hypocelluartity with increased temperature degrees and durations.

It is worth noting that the temperature during a bomb explosion ranges from 260°C to 293°C, whereas during a fire accident rises up to 500°C.[1] Although in this study Barr bodies were not detected beyond 200°C for 5-min duration, the conditions in this in vitro study is dissimilar to what occur in reality where the teeth are invested in bone and soft tissues that make the direct effect of heat less effective. Therefore, it is expected that the dental pulp tissue can endure higher temperature in vivo. Another limitation in this study is that negative or false positive results are obtained in female with chromosomes alteration as in aneuploid cases.[11] Therefore, this method for gender determination is not suitable in these cases.


  Conclusion Top


Dental pulp is considered an excellent tool for gender determination in various fire disasters and natural accidents as it is protected by mineralized dental tissues. Therefore, determination of dental Barr bodies may display a cardinal role in forensic situations when other evidences are not available especially in developing countries where the DNA analysis techniques might be slightly expensive. This study concluded that Barr bodies can be useful for gender determination up to 200°C at the time period of 5min in vitro. After that, its identification is not possible as degenerative changes occur in the pulp tissue. Further in vivo studies are recommended to determine the validity of using Barr body as a gender determining tool in the dental pulp tissue of females in heat exposure.

Ethical policy and institutional review board statement

Ethical approval for this study was obtained from the Research Ethics Committee of Faculty of Medicine, Tanta University (Protocol no. 330031/03/18).

Acknowledgement

We would like to thank Mohamed Elsaharty for sample collection and Dr. Fatma Mohamed Elgazzar for statistical analysis and interpretation of data.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
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.  Back to cited text no. 1
    
2.
Nayar A, Singh HP, Leekha S. Pulp tissue in sex determination: A fluorescent microscopic study. J Forensic Dent Sci 2014;6:77-80.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Pawar RK, More CB. Sex determination from tooth pulp deoxyribonucleic acid using polymerase chain reaction. J Forensic Dent Sci 2018;10:107-10.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Mânica S, Gorza L. Forensic odontology in the 21st century––identifying the opinions of those behind the teaching. J Forensic Leg Med 2019;64:7-13.  Back to cited text no. 4
    
5.
Krishan K, Kanchan T, Garg AK. Dental evidence in forensic identification––an overview, methodology and present status. Open Dent J 2015;9:250-6.  Back to cited text no. 5
    
6.
Gouri P, Kulkarni PG, Reddy P, Shyam D, Keerth M. Dental tissues as forensic tool in gender determination. Indian J Dent Adv 2016;8:37.  Back to cited text no. 6
    
7.
Barr ML, Carr DH. Sex chromatin, sex chromosomes and sex anomalies. Can Med Assoc J 1960;83:979-86.  Back to cited text no. 7
    
8.
Das N, Gorea RK, Gargi J, Singh JR. Sex determination from pulpal tissue. J Indian Acad Forensic Med 2004;26:50-54.  Back to cited text no. 8
    
9.
Suazo GI, Flores A, Roa I, Cantín M, Zavando D. Sex determination by observation of Barr body in teeth subjected to high temperatures. Int J Morphol 2011;29:199-203.  Back to cited text no. 9
    
10.
Suazo GI, Roa I, Cantín M. Sex chromatin in dental pulp: Performance of diagnosis test and gold standard generation. Int J Morphol 2010;28:1093-6.  Back to cited text no. 10
    
11.
Dawson-Saunders B, Trapp R. Basic and clinical biostatics. 3rd ed. Boston: Lange Medical Book/McGraw-Hill Medical Publishing Division;2001:p. 161–218.  Back to cited text no. 11
    
12.
Symes SA, Dirkmaat D, Ousley S, Chapman E, Cabo L. Recovery and interpretation of burned human remains. Final Technical Report Award. Washington, DC: National Institute of Justice, 2012 March; Report No: 237966, Award No: 2008-DNBX-K131.  Back to cited text no. 12
    
13.
Duffy JB, Waterfield JD, Skinner MF. Isolation of tooth pulp cells for sex chromatin studies in experimental dehydrated and cremated remains. Forensic Sci Int 1991;49:127-41.  Back to cited text no. 13
    
14.
Nanci A. Ten Cate’s Oral histology, Development, structure, and function. 9th ed. St. Louis, Missouri: Elsevier;2018:210–15.  Back to cited text no. 14
    
15.
Amin R, Shetty P, shetty V. Reliability of teeth for identification after exposure to varying degrees of temperature. World J Dent 2017;8:96-103.  Back to cited text no. 15
    
16.
Khanna S. Efficacy of sex determination from human dental pulp tissue and its reliability as a tool in forensic dentistry. J Int Oral Health 2015;7:10-16.  Back to cited text no. 16
    
17.
Yunis JJ, Chandler ME. Medical microscopy and other body fluids. In: Henry JB,editor. Clinical Diagnosis and Management Laboratory Methods. 16th ed. Philadelphia (PA): WB Saunders; 1979. p. 820-56.  Back to cited text no. 17
    
18.
Pericleous K, Bojarevics V, Djambazov G. Maximising heat transfer efficiency in the cold crucible induction melting process numerical heat transfer, Eds.: Nowak A, Białecki RA, Poland: Gliwice-Cracow; 2005.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed183    
    Printed4    
    Emailed0    
    PDF Downloaded16    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]