Aceto-orcein squash technique for barr body detection

INTRODUCTION

Forensic science is the application of technology to the criminal and civil laws that are enforced by police agencies in a criminal justice system.¹ The science applied in criminal cases is called “criminalistics”, which isconcerned with the recognition, identification, individualization and evaluation of physical evidence in matters of legal significance. Bite marks, blood and body fluids, bones, broken fingernails, drugs, fingerprints, glass, hair, and teeth are some of the materials which are considered as evidence.² Gender determination is considered one of the first and most important stepsin forensic medicine.³

Sex determination system is a biological system that determines the development of sexual characteristics in an organism. The determination of age and sex of an unknown body can be crucial to the investigators to limit the search for individuals that could possibly match for missing person. This can be achieved by several methods like deoxyribonucleic acid (DNA),polymerase chain reaction (PCR) and karyotyping which are expensive and not available in all places.Similarly, sex chromatin expressed by somatic cells can be used for sex determination.^3,4

In humans, there are 23 pairs of chromosomes. These chromosomes are present in the nucleus of every somatic cell. Each chromosome consists of DNA which is tightly wound and folded on a protein core.The first 22 pairs are called the autosomes.The 23rd pairis the sex chromosomesthe X and Y chromosomes.⁵ In females, the sex chromosomes are XX wherein one X is of paternal origin and other X is of maternal origin. Similarly, in males they are XY chromosomes. Among the two XX chromosomes in females, one X is active (Xa) and the other is in inactivate (Xi) state within the nucleus of female cells. The process by which one of the two copies of the X chromosome is inactivated is called “Lyonization”. The choice of X chromosome for lyonization is random. It can be either paternal or maternal X chromosome.⁶

The X chromosome selected for inactivation possess X-inactivation centre (XIC) which is lacking in Xa. The XIC contains four non-translated RNA genes -Xist, Tsix, Jpx and Ftx, which are involved in X-inactivation. Once a X chromosome is inactivated, it will remain inactive throughout the lifetime of the cell and its descendants, independent of the age of an individual⁷ The inactive X chromosome is silenced by it being packaged in such a way that it has a transcriptionally inactive structure called heterochromatin.6 DNA packaged in heterochromatin, such as the Xi, is more condensed than DNA packaged in euchromatin, such as the Xa. The inactive X forms a discrete body within the nucleus called “Barr Body”. The Barr body is generally located on the periphery of the nucleus of cells seen in the interphase of the cell cycle.⁸ Studies of individuals with extra copies of the X chromosome show that in cells with more than two X chromosomes, X inactivation and Barr body formation occurs according to the N- 1 rule: cells maintain a single Xa chromosome and condense all remaining X chromosomes into inactive state.⁹

During the course of investigation into the effects of stimulationon the microscopic appearance of the nerve cell of female cats, Barr and Bertram determined a small chromatin condensation within the nuclei – thus named it “Barr body”.¹⁰ The Barr body generally has a plano-convex, biconvex, triangular, spherical or rectangular morphology when observed in light microscopy. It resembles the letter V, W, S or X under electron microscope. It measures 0.8 – 1.1 micron in its greatest diameter.¹¹ Barr body has also been found in osteoblasts, osteocytes and periosteal cells of female dogs and cats (Vernino&Laskin, 1960),¹² cone cells of the human retina (Teplitz, 1965),¹³ mucosal cells (Dixon & Torr, 1956)^3,14 and on female calico cats.¹⁵

Barr chromatin can be observed with most of the nuclear stains, such as hematoxylin-eosin, Papanicolaou (PAP), Feulgen, cresyl violet, acetoorcein, carbol-fuchsin, and fluorescence (Duffy, 1991).¹⁶ PAP stain is a multi-chromatic staining cytologicaltechnique developed by George Papanicolaou in 1941. This polychrome staining method depends on degree of the cell maturity and cellular metabolicactivity.¹⁷

Theoral-mucosal-smear method (Moore and Barr 1955)¹⁸ can be used as a method for gender determination because smear may be obtained with minimal inconvenienceto the patient. The present paper describes the applicationof the acetoorcein squash technique to oralmucosal smears. This method of gender determinationis as accurate as other methods. Aceto-orcein technique has the additionaladvantage of revealing fine details of nuclear structures due to squashing and flattening the freshlyobtained cells.¹⁹

This study was carried out with an aim to identify ‘Barr bodies’ in human somatic cells, for gender determination. The efficacy of aceto-orcein and PAP stain was compared for detection of Barr bodies in buccal smears.

MATERIALS & METHODS

For this cohort analytic study the ethical approval was given by the ethical committee of our institution. Buccal smears were prepared from 250 healthy individuals who were selected within the age range of 19-28 years, from the out-patient department of our institution over a period of three months for the present study. The subjects who were healthy and had come for routine dental visit were included as samples for the study. Patients with any noticeable lesion in oral cavity and/or systemic disease were excluded. Among them, 125 were males and 125 were females. Two oral mucosal smears were prepared for each individual after taking his/her verbal consent. These smears were obtained by scraping the buccal mucosa of the individual with a cytology brush. It was rotated with firm pressure; the desquamated cells were then scooped up and spread on the surface of a clean dry slide.(Fig 1) One of these smears was stained with 1% aceto-orcein and the other with PAP stain.One drop of stain was quickly placed on the centre of the smear and a thin coverslip was laid over it. One or more layers of filter paper were placed on top of the coverslip and pressure was applied. The filter paper absorbs excess stain and the pressure applied causes even flattening of nuclei.¹⁹ The 1% aceto-orcein stain was prepared using synthetic orcein (Sisco Research Laboratories Pvt. Ltd, Mumbai) and glacial acetic acid according to the Sanderson method.^19,20 PAP stain was used to compare the efficacy of aceto-orcein. The staining of PAP was done according to the instruction manual available with the commercial preparation (Rapid PAP, Biolab Diagnostics, Jaipur.

The pathologist randomly analyzed 100 cells in each sample under microscope (Olympus CX21i) oil immersion (1000magnification) for identification of Barr bodies.

Inclusion criteria

The nuclei in which the Barr body was connected to a nucleolus by a fine chromatin thread were included in the study. The Barr body may appear in different shapes like flattened, convex or tentshaped at the nuclear membrane.¹⁹ (Fig 2).Extruded nuclei from the cell were also considered, provided the nuclear membrane remained intact.

Exclusion criteria

A pyknotic, degenerating, crumpled and overlapped nuclei were excluded from the study. Barr bodies may also lie in the centre of the nucleus, but cannot be distinguished from a nucleolus. Such centrally placed chromatin bodies were eliminated (Fig 3). Folded cells in which the cell membrane lies over the nucleus were not considered.¹⁹

Statistical Analysis

t-test was done for comparing the variance between identified barr bodies in two stains by using the calculated mean and standard deviation. The statistical significance was calculated < P 0.05As the sample size was not determined at the beginning of the study, based on results a power analysis was done.

RESULTS

The slides prepared were analysed for Barr body detection (Table 1). Barr bodies were not seen in any of the smears of males, but were identified in smears of females which were stained both with aceto-orcein and PAP. (Fig 4) For the statistical analysis, mean and standard deviation was calculated for identified Barr bodies in aceto-orcein and PAP stained slides of females. T-test was applied to compare the variance between aceto-orcein and PAP. A p-value below 0.05 was considered significant (Table 2). The difference in the number of Barr bodies identified between aceto-orcein smears and PAP stained smears in females was statistically significantat p<0.05. (Graph 1) The power analysis was done based on the results showing a significance of more than 80%.

DISCUSSION

Sex determination of unknown individuals is not always a straightforward procedure, especially when confronted by badly decomposed or severely traumatized remains.²⁰ This becomes even more challenging when very little remains of an individual are available as ante-mortem medical and/or dental records which may be inadequate, unavailable, or difficult to locate. Thus, any technique that is able to facilitate a rapid, simple, and inexpensive determination of sex is extremely important.²¹

There are large numbers of techniques designed for the purpose of sex determination. Diagnosing the sex of a biological evidence can provide important information in a forensic investigation. Many methods for determining the human males have been developed basically with the use of chromosome Y-specific sequences. On the other hand, there are few methods for the identification of females due to the lack of female-specific sequence.²²

Studies have been conducted since 1949 when Barr body was first demonstrated, to investigate the concept of its formation. In 1959, Susumu Ohno showed that the two X-chromosomes of mammals were different: one appeared like the autosomes; the other was condensed and heterochromatic. This finding suggested that one of the X-chromosomes underwent inactivation.²³ The fine chromatin thread which apparentlyconnects the sex-chromatin body to a nucleolus was firstrecognized by Reitalu (1957) in foetal tissue, and was subsequently described by Serr(1958) and his co-workers. Miles in1960 recognisedthis structure in oral mucosal smears.¹⁹

In 1961, Mary Lyon proposed the random inactivation of female X chromosome. Although the number of chromosomes might differ in some circumstances, there will be only one Xa chromosome and the Barr bodies might differ in number. But the number of Barr body is always one less than the number of X chromosomes existing in particular individual.^6,7 The following conditions will explain this phenomenon - Barr body number: XX - 1 Barr body; XXX -2 Barr bodies; XXY - 1 Barr body; XYY - no Barr body; XY - no Barr body.⁹

Other than gender determination in medico legal cases, the Barr body identification method has been of assistance in the field of sports. There were instances where male athletes posed as female athletes giving them an unfair advantage to win the competition. As a result, in 1968, the International Olympic Committee (IOC) at Mexico City Olympic Gamesadopted sex chromatin testing as an objective measure for gender verification.²⁴

The EMBO 50-years of X-inactivation conference in Oxford on July 22, 2011 incorporated the Lyon hypothesis that was proposed in 1961, as the Lyon Law.²⁵

Orcein preparations may be made with extreme ease and smears are stained rapidly within a minute.²⁰ Squashing of the nucleus, with subsequent enlargement, permits study of its finestructure.¹⁹ The performance in our analysis was 100%, makingit comparable to more complex techniques in identifying sex from amplification of DNA, which has a high specificity and sensitivity but require more complexwork and time to complete the analysis and need special equipment to amplify DNA.³ This technique has an advantage of ease of preparation, rapidity, revealing of fine details and are reliable.^11,19

The limitations to the use of this method are thealterations at the chromosomal level in conditions like Klinefelter’s syndrome 47 chromosomes (XXY), 48 chromosomes (XXXY), 49 chromosomes (XXXXY) in males, and triple-X (XXX) females. The nuclei of males with Klinefelter’s syndrome show presence of various numbers of barr bodies giving a false positive result.²⁶ Two barr bodies are seen in the nuclei of triple-X females, whereas only one barr body is seen in normal females.^3,18 Presence of two barr bodies can cause diagnostic dilemma as it is difficult to differentiate between a triple-X female and a variant of Klinefelter’s syndrome XXXY.⁹

The results of the presentstudy were similar to that of survey done by Anoop URet al¹¹ and Sanderson et al¹⁹ This study was done to analyse the diagnostic value of observation of sex chromatin in cells of buccal smears. Other than buccal smears, Barr bodies have also been isolated in cells of dental pulp tissue. In a study done in 2010, pulp tissue was extirpated from healthy teeth which were extracted for orthodontic treatment.²⁷ In blood smears Barr bodies can be seen in polymorphonuclear neutrophils. The Barr body in these cells appears more like a neutrophil appendage rather than a deposit of heterochromatin against the nuclear envelope. Barr body is evident in only 3% of neutrophils in females.²⁸

Astudy conducted on hair root sheath cells also observed sex chromatin. Hair samples were collected from dead bodies and stored in plastic bags. Later the hair was placed on the slide and the bulb of hair was cut using blade. The root sheath obtained was stained with aceto orcein and examined under oil immersion. There isaverage fall in percentage of Barr bodies with the passage of time due to the start of decompositionchanges in the root sheath cells. As hair is one of the trace evidences at the crime scene it can also be used for gender determination in forensic medicine.²⁹

Khorate MM et.al, has done a study on pulp tissue of extracted teeth. The stain used in their study was 5% quinacrine dihydrochloride and H&E for assessment of presence and absence of X & Y chromosomes. The conclusion of the study proposed that Barr body identification was a reliable method for gender determination.³⁰ In an anti-ubiquitin immunohistochemistry study by Ellen Gelpi on hippocampal granule cells in human brain for sex determination has mentioned that Barr body identification is a reliable method.³¹

CONCLUSION:

With the advancements in technology, DNA analysis is considered to be the most accurate method for human identification. But in the area of forensic science, where time and money are important parameters, Barr body identification for determining gender is a quick, easy, cost effective and reliable method. It is a simple method and can be readily repeated if necessary.

In the present study, aceto-orcein proved to be a more efficacious technique for gender determination by observing sex chromatin when compared with the conventional PAP method.