the main attributes of biological identity are sex , age , stature , and ethnic background of the individual , which are also called the  big four in forensic context . human identification is of paramount importance , for both legal as well as humanitarian purpose . albeit there have been tremendous advancements in forensic medicine , this remains a practical problem . the use of dental identification has long been considered a reliable method , especially when other methods ( fingerprints ) fail because body conditions are not available , and over time , forensic odontology has emerged as a complete branch . teeth can survive and remain virtually unaffected long after other soft tissue and skeletal tissues have been destroyed . the recognition of teeth as a tissue that withstands great variation in environment ( temperature upto 1600 c , humidity , and ph ) has lead to its application in personal identification . it has been observed in past time that dental tissues have witnessed its importance worldwide where it has contributed enormously in identification of victims in disaster , 80% of non - thai in tsunami in thailand and 20% in 26/11 incidence where 12 coordinated shooting and bombing attacks were carried by terrorists in mumbai , india 's most populous city on 26 november , 2010 were identified using teeth . other than this , it has been reported that renowned people like adolf hitler and indian prime minister rajiv gandhi were identified using tooth . several parameters have been proposed for sex determination to identify an individual from teeth , but none of them have proved its reliability . recently , dna analysis in forensic have gained attraction , but it is expensive and time - consuming . this prompted us to look for other methods of dental identification , which will be cost - effective yet reliable . tooth pulp is encased in a hard tissue casting , where it may be protected from detrimental effects of impact , trauma , and heat . developed a technique using pulpal tissue stained with quinacrine mustard , specific for y chromosome to determine sex of an individual . in literature , there is scarcity of relevant studies that analyzed the effect of caries on viability of pulpal tissue in sex determination . so , keeping in mind this fact , present study was conducted to analyze the effect of caries on pulpal tissue stained with quinacrine hydrochloride with the application of fluorescent microscope . this study was carried out in the department of oral pathology and microbiology , swami devi dyal hospital and dental college barwala , panchkula . the study protocol was approved by the ethical committee of college before commencement of the procedure . fifty maxillary and mandibular permanent teeth that were indicated for extraction for periodontal and endodontic reasons were included in the study . study sample consisted of 25 male and 25 female teeth , which were further categorized into 5 groups of 10 each ( 10 from males and 10 from females ) based on the extent of caries progression . group a includes freshly extracted tooth specimens with no caries , group b consisted of tooth specimens with caries in enamel , group c comprised of tooth specimens with caries less than half way of dentin , group d consisted of freshly extracted teeth with caries more than half way of dentin , and group e includes freshly extracted tooth specimen with caries involving pulp . the teeth were examined radiographically and clinically for analyzing the various stages of caries progression [ figure 1 ] . ( a ) sound teeth , ( b ) enamel , ( c ) less than half way of dentin , ( d ) more than half way of dentin , ( e ) pulp modeling wax was folded and made into a block . the tooth was embedded on the modeling block . to remove the pulp , the crown it was adequately washed in normal saline to remove any calcified bone or dentine particles . the pulp tissue was then transferred to the dry and clean conical centrifuge tubes containing 5 ml of fixative ( 3 methanol:1 glacial acetic acid ) and left as such for about half an hour to 2 hours for the fixation of the pulp cells . it was then crushed with the glass rod sufficiently to isolate the pulp cells . a suspension five ml of fresh fixative was then added to re - suspend the pellet , and the process was repeated thrice till a clear suspension of the pulp cells was obtained . thin smears were prepared on chilled microscope slides of 1 mm thickness by the air - drying method i.e. , by dropping 2 - 3 drops of the above suspension on the slide from a distance of inches to get a homogenous population of cells . the slide was mounted in buffer of ph 5.5 and was observed under leica dmr fluorescent microscope ( oil immersion in dark field at 40 ) by bv exciting method ( emitting a blue - violet color , mainly at 4.047a0 and 4.038a0 ) . only those cells which contained the characteristic y chromatin i.e. , a brightly fluorescent spot attached to the nuclear membrane were counted as positive cells while those which did not show any such fluorescent spot were labeled as negative . results obtained were subjected to statistical analysis by using independent t - test using spss ( version 9.0 ) software . the tooth was embedded on the modeling block . to remove the pulp , the crown it was adequately washed in normal saline to remove any calcified bone or dentine particles . the pulp tissue was then transferred to the dry and clean conical centrifuge tubes containing 5 ml of fixative ( 3 methanol:1 glacial acetic acid ) and left as such for about half an hour to 2 hours for the fixation of the pulp cells . it was then crushed with the glass rod sufficiently to isolate the pulp cells . a suspension five ml of fresh fixative was then added to re - suspend the pellet , and the process was repeated thrice till a clear suspension of the pulp cells was obtained . thin smears were prepared on chilled microscope slides of 1 mm thickness by the air - drying method i.e. , by dropping 2 - 3 drops of the above suspension on the slide from a distance of inches to get a homogenous population of cells . the slide was mounted in buffer of ph 5.5 and was observed under leica dmr fluorescent microscope ( oil immersion in dark field at 40 ) by bv exciting method ( emitting a blue - violet color , mainly at 4.047a0 and 4.038a0 ) . only those cells which contained the characteristic y chromatin i.e. , a brightly fluorescent spot attached to the nuclear membrane were counted as positive cells while those which did not show any such fluorescent spot were labeled as negative . results obtained were subjected to statistical analysis by using independent t - test using spss ( version 9.0 ) software . this study was done to evaluate the use of the fluorescent body test as a diagnostic test for sex determination . table 1 showed sensitivity , specificity , positive predictive value , negative predictive value , and efficiency of each of the five groups . the values obtained were highest for sound teeth with caries in enamel and value decreases with the progression of the caries . table 2 depicted that fluorescent bodies were found to be more in sound teeth in males as the caries increase , the mean percentage of fluorescent bodies observed decreases in males . we also observed the fluorescent spots in females , and the value of the spot increases in female as the caries progresses , thereby giving false positive results in females [ figure 3 ] . the p value were found to be significant for sound teeth and teeth with caries in enamel and involving less than half the way of dentin , whereas it was non - significant for teeth involved with further caries involvement . diagnostic utility of fluorescent body in identifying gender percentage of fluorescent bodies ( in % ) in different groups fluorescent photomicrograph showing bright fluorescent cell in males representing y chromosome fluorescent photomicrograph representing faint fluorescence in females despite the fact that tremendous research work is carried out to determine a reliable method , but none have proven its efficacy . the most reliable means of identification include fingerprints , dental comparisons , and biologic methods such as dna profiling . in some cases , however , fingerprints are not available from the deceased , or ante - mortem prints can not be obtained . sex determination from skull is highly unreliable although number of workers have described differences in the size of male and female teeth , such measurements seem unlikely to be used to determine the sex of an individual . so , a technique has been developed , which may offer a solution whenever soft tissue remains can be recovered . casperson et al . showed that chromosome stained with quinacrine mustard fluoresced differentially along their length when viewed in ultraviolet light and the human y chromosome fluoresced more brightly than other chromosomes . he suggested that alkylating agents like quinacrine acts on the dna portion rich in guanine and accumulate there . this technique has been used in forensic science for sex determination from dried blood stains , saliva , and hair . thus , it might be applicable to sex determination from tooth pulp some time after death , because the teeth are a stable part of the skeleton and the pulp tissue is well - protected . seno and ishizu carried out the detection of y chromosome in the nuclei of dental pulp . they found that f- body positive rate in the nucleated cells of male dental pulp was over 30% , even with the male teeth as old as 5 months , after the extraction . with female teeth , typical f - body can not be detected , and f - body like spot has been observed in 0.4% of cells , indicating that there can be no error in the identification of male tooth from that of female one , even such an f - body like spot is taken as an f - body itself . present study revealed that in males , 60 - 75% fluorescent y bodies [ figure 2 ] were observed in sound teeth ( mean 69.4% bodies ) , 59 - 70% bodies in tooth with caries in enamel ( mean 64.2% bodies ) , 19 - 53% in teeth with caries in half way of dentin ( mean 39.8% bodies ) , teeth with caries involving more than half way of dentin and involving pulp showed 13 - 29% , 1 - 19% ( mean 21.8% , 6.37% bodies ) off bodies respectively as shown in table 2 . seno and ishizu showed a range of 56 - 72% fluorescent y bodies in freshly extracted sound teeth , which are in accordance to sound teeth in this study . pseudo y body in the present study [ figure 4 ] in females were found to be in the range of 0 - 12% ( mean 5.2% ) , 0 - 12% ( mean 5.2% ) , 14 - 22% ( mean 17.8)% , 12 - 22 ( mean 16.8% ) , and 7 - 15% ( mean 11.6% ) for sound teeth , teeth with caries in enamel , caries in half way of dentin , caries involving more than half way of dentin caries , and caries involving pulp , respectively , as shown in table 2 . fluorescent photomicrograph representing artifacts or pseudo f - bodies in females discrepancy between various studies could be attributed to random inclusion of carious and non - carious teeth . so , the present study considered the effect of caries on fluorescent body test as literature ( when searched via pubmed and medline ) revealed lack of studies analyzing this fact . from present study , it can be inferred that as the caries progresses from enamel to dentin and further to pulp tissue , the sensitivity and specificity of determining y chromosome decreases [ table 1 ] . when the caries have involved only enamel or half the way of dentin , it has shown significant difference in fluorescent body test determining males and females , unlike when it involved more than half way of dentin and pulp where the difference is non - significant [ table 2 ] . the decrease in  y body could be attributed to the fact that cariogenic bacteria alter the microenvironment of pulp , resulting in alteration in fluorescent staining . as the caries progresses , the inflammatory processes in the pulp are initiated , as the inflammation progresses the internal architecture of the cell is lost , and is also characterized by necrotic poorly stained cellular debris . many workers attributed the appearance of fluorescent bodies in females to artifacts , whereas others ascribed it to the presence of fluorescent debris . it was necessary to smear pulp cells thinly to prevent masking the fluorescent y chromosomes by fluorescent debris . sex determination by fluorescent staining of the y chromosome is a reliable technique in teeth with healthy pulp tissue or caries with enamel or upto half way of dentin .
aims : to determine and compare the reliability of pulp tissue in determination of sex and to analyze whether caries have any effect on fluorescent body test.materials and methods : this study was carried on 50 maxillary and mandibular teeth ( 25 male teeth and 25 female teeth ) , which were indicated for extraction . 
 the teeth are categorized into 5 groups , 10 each ( 5 from males and 5 from females ) on the basis of caries progression . 
 the pulp cells are stained with quinacrine hydrochloride and observed with fluorescent microscope for fluorescent body . 
 gender is determined by identification of y chromosome fluorescence in dental pulp.results:fluorescent bodies were found to be more in sound teeth in males as the caries increase the mean percentage of fluorescent bodies observed decreases in males . 
 we also observed the fluorescent spots in females , and the value of the spot increases in female as the caries progresses , thereby giving false positive results in females.conclusion:sex determination by fluorescent staining of the y chromosome is a reliable technique in teeth with healthy pulps or caries with enamel or up to half way of dentin . 
 teeth with caries involving pulp can not be used for sex determination .