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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 1  |  Page : 7-10

Efficacy of acridine orange and papanicolaou stains in sex determination using barr bodies in buccal smears: A comparative study


1 Assistant Professor, Department of Oral Pathology, Century International Institute of Dental Sciences, Poinachi, Kerala, India
2 Reader, Department of Oral Pathology, AJ Institute of Dental Sciences, Mangalore, Karnataka, India
3 Reader, Department of Oral Pathology, SJM Dental College and Hospital, Chitradurga, India
4 Assistant Professor, Department of Prosthodontics, Century Institute of Dental Sciences, Kasargod, Kerala, India

Date of Web Publication26-Jul-2019

Correspondence Address:
Dr. Frankantony P Britto
Department of Oral Pathology, SJM Dental College and Hospital, Chitradurga - 577 501, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/INPC.INPC_11_19

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  Abstract 


Objective: Sex determination can be done by buccal epithelial cells in saliva traces found at a crime scene by examining the presence of Barr bodies in the nucleus of the epithelial cells. The present study aims to assess the efficacy of sex determination using Acridine Orange (AO) and Papanicolaou (PAP) stains for the detection of Barr bodies in buccal mucosal scrapings.
Materials and Methods: Buccal scrapings from 120 healthy individuals (60 males and 60 females) were collected and were stained with AO and PAP stains. The analysis of fifty epithelial cells in each sample was done for the identification of Barr bodies. The presence of Barr bodies ≤ 5% in the sample was recorded as male and those with > 5% was recorded as female. Both stains were evaluated for percentage accuracy in determining sex.
Results: In AO-stained slides, the percentage of Barr bodies ranged from 4 to 31 in females and from 0 to 9 in males, whereas with PAP the ranges recorded were 3–21 in females and 0–6 in males (P < 0.001). AO and PAP stains for detecting sex accurately, showed sensitivity and specificity of around 97.9% and 96.2%, respectively.
Conclusion: Using Barr bodies in the buccal cells, providing up to 95%–98% accuracy, made it a considerable aid for sex determination. AO stain proved better than PAP stain for visualizing nuclear details with its remarkably shorter staining time and confirmed superior sex estimation efficiency compared to PAP stain.

Keywords: Acridine orange, Barr bodies, buccal scrapings, Papanicolaou


How to cite this article:
Aziz NZ, Prasad B G, Arathi K, Britto FP, Chethan Aradhya B V, Abdulrashid B. Efficacy of acridine orange and papanicolaou stains in sex determination using barr bodies in buccal smears: A comparative study. Int J Prev Clin Dent Res 2019;6:7-10

How to cite this URL:
Aziz NZ, Prasad B G, Arathi K, Britto FP, Chethan Aradhya B V, Abdulrashid B. Efficacy of acridine orange and papanicolaou stains in sex determination using barr bodies in buccal smears: A comparative study. Int J Prev Clin Dent Res [serial online] 2019 [cited 2019 Nov 17];6:7-10. Available from: http://www.ijpcdr.org/text.asp?2019/6/1/7/259165




  Introduction Top


Forensic science plays a vital role in the establishment of the identity of an individual, and sex determination is the initial step in personal identification. Sex determination can be done by obtaining blood, semen, hair, and saliva stains containing buccal mucosal cells found in human body or at the disaster sites.[1]

Barr and Bertram in 1949first described the sex chromatin in mammals. This sex chromatin/Barr body is typically found in female nuclei. These Barr bodies are Feulgen positive, heteropyknotic, basophilic, intranuclear intensely stained structures. They are found inside the nuclear membrane, at the periphery of the nucleus being plano-convex or triangular in shape with a diameter of 1 μm.[2]

The outcome of the gene product is similar in both males and females even if females have 2X chromosomes (XX) and males (XY) have only 1X chromosome.  Lyonization More Details was explained by Lyon wherein the process of inactivation of one of the X chromosomes of the females takes place in each somatic cell, during early female embryonic development.[3],[4] Barr bodies also fluoresce because they are nuclear structures and all nuclear structures are known to fluoresce.[5] Barr bodies can be confirmed by various stains such as hematoxylin and eosin, Papanicolaou (PAP), orcein, cresyl violet, carbol fuchsin, Feulgen guard, and fluorescent stains.[6],[7]

The present study was conducted to compare and evaluate the efficacy of acridine orange (AO) and PAP stains in sex determination using Barr bodies in buccal smears under a fluorescent microscope.


  Materials and Methods Top


After obtaining institutional ethical committee clearance, informed consent was obtained from the participants. Two smears each were collected from 120 healthy individuals (60 males and 60 females), the total sample size accounting to 240. Healthy individuals were preferred in the study. Exclusion criteria included patients with known oral mucosal lesions, systemic illness, and deleterious habits such as tobacco chewing. Preprocedural rinsing of mouth was done by the patients with mouthwash solution followed by water. Using a sterilized metal spatula, the buccal mucosa was scrapped and the cellular material was collected. The first scraping was discarded owing to contamination with food debris and mucous. Using a fresh spatula, the second scrapping was done to collect cells from cleaner deep epithelial layers. The collected scraping was then transferred onto two clean slides making two smears per individual. The obtained smears were prepared by air drying and fixed in 95% isopropyl alcohol for 30 min before staining. The smears collected were stained with both PAP and AO stains.

The PAP-stained smears and AO-stained smears were observed under an oil immersion microscope and fluorescent microscope, respectively. Fifty cells were observed on each slide, and the total number of Barr body-positive cells was counted. The number of Barr body-positive cells was compared in both males and females by observing smears stained with PAP and with AO stains.

The statistical methods of standard deviation and arithmetic mean were used, and the data collected were compared with the reference values given for both genders with regard to the prevalence of Barr body-positive cells. The efficiency of both PAP and AO stains was compared using Mann–Whitney U-test.


  Results Top


One hundred and twenty individuals participated in the study (60 males and 60 females; age range, 20–40 years). On examination of AO-stained slides, the percentage of Barr bodies was found to be ranging from 4 to 31 in females (mean 9.63 ± 3.76) [Figure 1] and 0 to 9 (mean 2.78 ± 0.89) in males [Figure 2]. In PAP-stained smears, the percentage of Barr body ranged from 3 to 21 in females (mean 10.96 ± 4.38) [Figure 3] and 0 to 6 in males (mean 2.37 ± 1.12) [Figure 4]. The Mann–Whitney test showed significant differences (P < 0.001) in studying the relation of Barr body percentage in male and female patients. Both AO and PAP stains showed superior intra-and interobserver correlation. Further, the sensitivity and specificity of AO for detecting sex accurately was found to be 97%, with an overall accuracy of 97.9%, whereas for PAP the sensitivity was 91% and the specificity was 99%, with an overall accuracy of 94.2%. AO was considerably better in the sex estimation efficacy in females [Table 1].
Figure 1: Acridine orange-stained smear showing Barr body in a female buccal smear

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Figure 2: Acridine orange-stained smear of a male buccal smear

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Figure 3: Papanicolaou-stained smear showing Barr body in a female buccal smear

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Figure 4: Papanicolaou-stained smear showing Barr body in a male buccal smear

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Table 1: Sensitivity and specificity of Papanicolaou and acridine orange for sex determination

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  Discussion Top


Sex determination is the first step in forensic identification, and expression of nuclear sex chromatin plays a fundamental role as far as sex determination in individuals is concerned.[8] In normal males and females, the chromatin pattern is different for both. Sex chromatin is a unique feature of the female nuclei, being used in the past to typify normal females.[1],[9],[10] The presence or absence of Barr bodies in females can depend on variety of factors such as age, gender, pregnancy, menstruation cycle, and chromosomal abnormalities such as Klienfelters syndrome and Down syndrome.[11],[12],[13] There was marked variation in the percentage of Barr bodies in premalignant and malignant lesions.[14],[15] Cytological smears and staining techniques are cost-effective and feasible for sex determination. Various stains are used to reveal Barr bodies – PAP, Feulgen, cresyl violet, orcein, carbol fuchsin, and fluorescent staining.[7],[16] The purpose of using AO and PAP in the current study is due to its time-effectiveness, cost-effectiveness, and relative simplicity.[17],[18] In the present study, the mean percentages of Barr bodies were consistently higher in females than in males, which is in concordance with other studies.

The present study correlated with the study done by Datar et al. in 2013, wherein the percentage of Barr bodies in AO-stained slides ranged from 5 to 32 in females and from 0 to 8 in males, whereas with PAP the ranges recorded were 4–20 in females and 0–5 in males (P < 0.001). In our study, in AO-stained slides, the percentage of Barr bodies ranged from 4 to 31 in females and from 0 to 9 in males, whereas with PAP the ranges recorded were 3–21 in females and 0–6 in males (P < 0.001). The present study showed sensitivity and specificity of around 97.9 and 96.2%, respectively, for AO and PAP stains used for detecting sex accurately, whereas the study by Datar et al. showed sensitivity and specificity of 98.3 and 95% for AO and PAP, respectively.[1]

Reddy et al. in their study using a small sample size of 20 males and females each observed a Barr body percentage of 18%–72%, and all the females expressed Barr bodies. Herein, the smears were stained with AO stain as in our study. Our study showed lesser Barr body percentage of 4%–31% cells possibly due to the variation in the microscopic observation technique. Our study used fluorescent microscope as against confocal microscope in the study done by Reddy et al., which yielded better results.[3]

Baby et al. carried out a study for the expression of Barr bodies using PAP and acriflavine Schiff (AF) staining methods. AF Schiff-stained positive cells ranged from 16% to 53% and PAP-stained positive cells ranged from 9% to 38% in females. In males, 0%–9% AF-positive Barr bodies and 0%–5% PAP-stained Barr bodies were recognized. However, in our study, in females, AO-stained positive cells ranged from 4% to 31% and PAP showed 3%–21%. In males, 0%–9% AO-positive Barr bodies and 0%–6% PAP-stained Barr bodies were recognized. The present study showed a better correlation with the above study using PAP stain.[19]

The present study depicted a higher mean percentage of Barr bodies in females as compared to males. AO confirmed a superior diagnostic precision for sex determination in comparison with PAP. AO has a greater benefit when accurate diagnosis is essential.


  Conclusion Top


The AO stain for Barr body is better than the usual PAP stain because it is more consistent with shorter staining time and better revelation of nuclear details, thereby signifying superior sex estimation efficiency.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Datar U, Angadi PV, Hallikerimath S, Kale AD. Cytological assessment of Barr bodies using aceto-orcein and Papanicolaou stains in buccal mucosal smears and their sex estimation efficacy in an Indian sample. Acta Cytol 2013;57:516-21.  Back to cited text no. 1
    
2.
Kaur N, Sidhu R, Chandra S, Taneja N. Buccal Barr bodies: Accuracy and reliability in sex determination. Saudi J Oral Dent Res 2017;2:168-73.  Back to cited text no. 2
    
3.
Reddy DS, Sherlin HJ, Ramani P, Prakash PA. Determination of sex by exfoliative cytology using acridine orange confocal microscopy: A short study. J Forensic Dent Sci 2012;4:66-9.  Back to cited text no. 3
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4.
Anoop UR, Ramesh V, Balamurali PD, Nirima O, Premalatha B, Karthikshree VP, et al. Role of Barr bodies obtained from oral smears in the determination of sex. Indian J Dent Res 2004;15:5-7.  Back to cited text no. 4
    
5.
Mittal T, Saralaya KM, Kuruvilla A, Achary C. Sex determination from buccal mucosa scrapes. Int J Legal Med 2009;123:437-40.  Back to cited text no. 5
    
6.
Priyadharscini RA, Sabarinath T. Barr bodies in sex determination. J Forensic Dent Sci 2013;5:64-7.  Back to cited text no. 6
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7.
Nagamori H, Ohno Y, Uchima E, Kajiwara M, Nakazato M, Une Y, et al. Sex determination from buccal mucosa and hair root by the combined treatment of quinacrine staining and the fluorescent Feulgen reaction using a single specimen. Forensic Sci Int 1986;31:119-28.  Back to cited text no. 7
    
8.
Galdames IS, Henriquez IR, Cantin 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. 8
    
9.
Oliveira AM, French CA. Applications of fluorescence in situ hybridization in cytopathology: A review. Acta Cytol 2005;49:587-94.  Back to cited text no. 9
    
10.
Aggarwal NK, Kumar S, Banerjee KK, Agarwal BB. Sex determination from buccal mucosa. J Forensic Med Toxicol 1996;13:43-4.  Back to cited text no. 10
    
11.
Gonzales HC, Mendes FT, Saldanha PH. Sexchromatin in normal newborns in first two weeks of life: A blind study. Rev Brazil Genet 1978;4:263-78.  Back to cited text no. 11
    
12.
Hagy GW, Brodrick MM. Variation of sex chromatin in human oral mucosa during menstrual cycle. Acta Cytol 1972;16:314-20.  Back to cited text no. 12
    
13.
Hamerton JL, Jagiello GM, Kirman BH. Sex-chromosome abnormalities in a population of mentally defective children. Br Med J 1962;1:220-3.  Back to cited text no. 13
    
14.
Ghosh SN, Shah PN, Desai PB. Barr body frequency in esophageal cancer in Indian women. Acta Cytol 1983;27:202-3.  Back to cited text no. 14
    
15.
Nishiya I, Ishizaki Y, Sasaki M. Nuclear DNA content and the number of Barr bodies in premalignant and malignant lesions of the uterine cervix. Acta Cytol 1981;25:407-11.  Back to cited text no. 15
    
16.
Bancroft JD, Stevens A. Theories and Practice of Histological Techniques. 4th ed. London: Churchill Livingstone;1996. p. 114-50.  Back to cited text no. 16
    
17.
Sanderson AR, Stewart JS. Nuclear sexing with acetoorcein. Br Med J 1961;2:1065-7.  Back to cited text no. 17
    
18.
Meena NK, Singh M, Singh J. A comparison study of carbol fuchsin and Papanicolaou staining methods for the demonstrationand enumeration of Barr bodies in buccal smear. Int J Appl Res 2016;2:941-4.  Back to cited text no. 18
    
19.
Baby TK, Thomas P, Palani J, Pillai RK, Ramakrishnan BP. Sex determination efficacy of Papanicolaou and acriflavine schiff stains in buccal smears. J Forensic Dent Sci 2017;9:46.  Back to cited text no. 19
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