Isolation of tooth pulp cells for sex chromatin studies in experimental dehydrated and cremated remains.

In experiments designed to assess sex chromatin in artificially mummified and heated pulp tissue, a method was devised that successfully separates cells while minimizing nuclear damage. Sex chromatin (both Barr bodies and F-bodies) is shown to preserve in dehydrated human pulps up to one year. Human pulp tissue retains sex diagnostic characteristics when heated to 100 degrees C for up to 1 h. Parallel experiments on extracted teeth from young pigs reveals comparable tissue preservation. Heat penetration is retarded, however, in unextracted pig teeth in fleshed jaws such that temperatures could be raised to 300 degrees C for longer than 1 h. Heat penetration into fleshed material was further tested by the insertion of thermocouple probes to assess the temperature attained within the pulp chamber. At chamber temperatures up to 75 degrees C sex diagnosis in human pulps from extracted teeth was still possible. In outdoor incineration of fleshed pigs' heads in an open fire, 75 degrees C in the pulp chamber was reached at a fire temperature within the range 500-700 degrees C. The implications of these findings for forensic situations are described.     

Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions.

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.     

Radiographic evaluation of teeth subjected to high temperatures: experimental study to aid identification processes

The radiographic evaluation of dental remains represents a significant aspect in the forensic identification process, particularly after an exposure to fire. The aim of this "in vitro" study was to evaluate the radiographic features of unrestored, endodontically treated and restored teeth after exposure to an experimental range of high temperatures. Ninety human teeth were divided into two groups: (1) unrestored teeth, as a control group and (2) teeth endodontically treated (condensation technique) and restored with amalgam or composite fillings. Before testing the high temperatures, periapical radiographs of all teeth were performed. The tests of exposure to heat were carried out in an oven for six different temperatures (200, 400, 600, 800, 1000 and 1100 degrees C (392, 752, 1112, 1472, 1832, 2012 degrees F)). After each exposure, periapical radiographs of all the teeth were taken. The radiographic appearance of all the teeth before and after the thermal stresses were evaluated and the differences were recorded. The results of the radiographic examination showed that a number of significant radiographic details were conserved: the composite fillings were in place maintaining the shape till 600 degrees C (1112 degrees F), the amalgam fillings were in place maintaining the shape till 1000 degrees C (1832 degrees F) and the endodontic treatments were recognisable till 1100 degrees C (2012 degrees F).     

Scanning electron microscopy of incinerated teeth

The aim of the present scanning electron microscopy study was to document the nature of morphologic changes occurring in human enamel and dentin subjected in vitro to temperatures in the range of 200-1,000 degrees C for variable times. The results of the investigation confirm that human enamel and dentin remain microscopically identifiable after incineration at 1000 degrees C. Furthermore, these tissues remain identifiable after incineration at 1,000 degrees C for periods greater than 3 h. No consistent or reliable differences in morphology could be detected in enamel or mineralized dentin incinerated in the temperature range 200-600 degrees C. Temperature-dependent changes involving the predentin zone were observed. Following incineration at 800 degrees C for over 3 h and at 1,000 degrees C for 3 h, a metamorphosis of enamel and dentin into a globular form was observed.     

Identification of incinerated root canal filling materials after exposure to high heat incineration

With the increase in global terrorism there is a higher probability of having to identify victims of incineration events secondary to incendiary explosive devices. The victims of incineration events challenge forensic odontologists when coronal restorations are no longer present to compile postmortem data. With 40 million root canals being completed annually in the United States, a very large pool of antemortem data is available to the forensic odontologist to make positive identifications. When complete and thorough dental records exist, individuals that have undergone surgical and nonsurgical root canal therapy may have materials present in the canal that may aid in identification. This study provides elemental fingerprints of root canal obturation materials to be utilized as a forensic identification aid. This study used scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) to assess the elemental composition of materials before and after high temperature incineration. Sixteen endodontic materials were analyzed pre-incineration and placed in extracted teeth. The filled teeth were subjected to incineration at 900 degrees C for 30 min to simulate incineration events or cremation. Incinerated materials were radiographed and re-analyzed to determine if they retained their original elemental composition. Endodontic sealers, gutta percha, root-end filling materials, silver points, and separated files were distinguishable in the canal and traceable after incineration. The authors present a fingerprint of the endodontic obturation materials that are capable of withstanding high heat incineration to be used as an aid for postmortem identification. This work represents the initial stage of database generation for root canal filling materials for use as an aid in forensic identification.     

Chelating resin-based extraction of DNA from dental pulp and sex determination from incinerated teeth with Y-chromosomal alphoid repeat and short tandem repeats

A procedure utilizing Chelex 100, chelating resin, was adapted to extract DNA from dental pulp. The procedure was simple and rapid, involved no organic solvents, and did not require multiple tube transfers. The extraction of DNA from dental pulp using this method was as efficient, or more so, than using proteinase K and phenol-chloroform extraction. In this study, the Chelex method was used with amplification and typing at Y-chromosomal loci to determine the effects of temperature on the sex determination of the teeth. The extracted teeth were incinerated in a dental furnace for 2 minutes at 100 degrees C, 200 degrees C, 300 degrees C, 400 degrees C, and 500 degrees C. After the isolation of DNA from the dental pulp by the Chelex method, alphoid repeats, and short tandem repeats, the human Y chromosome (DYZ3), DYS19, SYS389, DYS390, and DYS393 could be amplified and typed in all samples incinerated at up to 300 degrees C for 2 minutes. The DYS389 locus in some samples could not be amplified at 300 degrees C for 2 minutes. An autopsy case is described in which genotypings of DYS19, DYS390, and DYS393 from dental pulp obtained from a burned body were needed. The data presented in this report suggest that Chelex 100-based DNA extraction, amplification, and typing are possible in burned teeth in forensic autopsy cases.     

Detection and classification of composite resins in incinerated teeth for forensic purposes

The great demand for esthetic restorations has resulted in placement of large numbers of composite resin fillings. The popularity of these materials is reflected in the quantity and variety of resin brands currently on the market. The ability to distinguish resin brands can aid in positive identification of burn victims, assuming that appropriate dental records exist. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) was used to characterize the structure and composition of 10 modern resins. The structure of each resin was unique to manufacturer, and elemental analysis allowed separation into distinct groups. These 10 resins were also placed in extracted teeth and incinerated at 900 degrees C for 30 min, simulating near cremation conditions. The resins were identifiable by SEM/EDS after incineration, and the elemental composition remained almost unchanged. The data produced are immediately useful for resin identification in forensics, and comparative analysis can be readily performed using standard equipment. This work represents the initial stage of database generation.     

Analysis of 14C and 13C in teeth provides precise birth dating and clues to geographical origin

The identification of human bodies in situations when there are no clues as to the person's identity from circumstantial data, poses a difficult problem to the investigators. The determination of age and sex of the body can be crucial in order to limit the search to individuals that are a possible match. We analyzed the proportion of bomb pulse derived carbon-14 ((14)C) incorporated in the enamel of teeth from individuals from different geographical locations. The 'bomb pulse' refers to a significant increase in (14)C levels in the atmosphere caused by above ground test detonations of nuclear weapons during the cold war (1955-1963). By comparing (14)C levels in enamel with (14)C atmospheric levels systematically recorded over time, high precision birth dating of modern biological material is possible. Above ground nuclear bomb testing was largely restricted to a couple of locations in the northern hemisphere, producing differences in atmospheric (14)C levels at various geographical regions, particularly in the early phase. Therefore, we examined the precision of (14)C birth dating of enamel as a function of time of formation and geographical location. We also investigated the use of the stable isotope (13)C as an indicator of geographical origin of an individual. Dental enamel was isolated from 95 teeth extracted from 84 individuals to study the precision of the (14)C method along the bomb spike. For teeth formed before 1955 (N=17), all but one tooth showed negative Δ(14)C values. Analysis of enamel from teeth formed during the rising part of the bomb-spike (1955-1963, N=12) and after the peak (>1963, N=66) resulted in an average absolute date of birth estimation error of 1.9±1.4 and 1.3±1.0 years, respectively. Geographical location of an individual had no adverse effect on the precision of year of birth estimation using radiocarbon dating. In 46 teeth, measurement of (13)C was also performed. Scandinavian teeth showed a substantially greater depression in average δ(13)C (-14.8) than teeth from subjects raised in Japan (-13.5), Middle East and North Africa (-12.7) and South America (-10.9). In summary, isotopic analysis of carbon in enamel from a single tooth can give a good estimate of the year of birth of an individual and also provide information about the geographical origin of the individual. This strategy can assist police and forensic authorities when attempting to solve unidentified homicide cases and may facilitate the identification work associated with mass disasters.     

Fluorescence and Structural Degradation in Composite Resins as a Function of Temperature*

Abstract: Detecting composite resin upon postmortem examination can be difficult. Ultraviolet illumination has been suggested to ease location of this material; however, this may not be advisable in incineration situations. Understanding of the chemical and physical properties of resin as a function of temperature is an important parameter in identification of this material in incineration circumstances. Twenty-seven discs of resin, Quixx (Dentsply), Filtek Supreme (3 M), and Tetric Ceram (Ivoclar) were prepared and exposed to increasing heating conditions of 200°C–900°C in 100°C increments for 30 min. Analysis was performed with Fourier transform infrared spectroscopy, ultraviolet-visible light spectrophotometry, scanning electron microscopy/energy dispersive X-ray spectroscopy, optical microscopy, and UV illumination. Characterization of the material occurred at each temperature range. The organic components and the fluorescence properties were lost at temperatures above 300°C. The inorganic component remained through 900°C. This information can aid in detection of resin in high temperature circumstances.     

Development of a radioimmunoassay technique for the detection of human hemoglobin in dried bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.     

A standard procedure for accommodating forensic anthropological and genetic analysis of decomposing human remains from tropical climates

At the Medical Legal Center in Ribeirão Preto, Brazil (CEMEL/FMRP-USP), unidentified decomposing bodies routinely undergo soft tissue removal (by immersion in water at 80–90 °C for 24 h) prior to an anthropological analysis intended to yield a biological profile of age, sex, ancestry, height, pathology and so on. In the event that this analysis is unsuccessful, samples may be submitted for DNA profiling. The tropical climate and the defleshing process may confound preservation, recovery and analysis of DNA, however. In order to establish an optimal standardized protocol for identification of decomposing human remains from a tropical climatic region, the outcome of anthropological and genetic analyses was compared, along with the utility of bone (mainly femur and sternum) and teeth (mainly molar) specimens for DNA analysis. In a sample (n = 39) of partially skeletonized remains, anthropological analysis was sufficient for identification in eight cases. In further six cases, DNA profiling was successfully attempted. As a consequence of our study, we recommend collection of 1–2 well preserved teeth prior to defleshing and anthropological analysis in these circumstances.     

Heat Induced Changes to Dental Resin Composites: A Reference in Forensic Investigations?*

The objective was to investigate color change and surface damage in dental resin composites exposed to high temperatures over different time intervals for comparative purposes. Samples were prepared using two resins - Z100(R) (R1) and Charisma (R2), heated at the following temperatures: 200 degrees C, 400 degrees C, 600 degrees C, 1000 degrees C, for 15, 30 and 45 min (n = 104 for each resin sample). Color (DeltaE) and brightness (DeltaL) changes were analyzed by spectrophotometry using the CIE Lab system and surface changes by Scanning Electron Microscopy (SEM). R1 showed more intense color changes after heat exposure than R2. DeltaL values were found to be the best parameter for evaluation of light and color change. A biphasic pattern after thermal exposure was detected, from dark brown to light white. SEM showed more intense alterations in R2 than in R1. These results indicate that the parameters observed in both resins are useful as a guide in forensic analyses.     

The heat resistance of a data-encoded ceramic microchip identification system

Samis introduced the concept of the Dentify system of personal identification. In this system, ceramic microchips embossed with metallic intelligence data are placed in teeth under dental restorations. This paper describes the results of an in vitro study aimed at testing the heat resistance qualities of this identification system when placed in teeth. Teeth were subjected to a temperature of 1,000 degrees C. The results of this study indicate that the ceramic chips and their embossed metallic intelligence data are able to withstand extremely high temperatures. The metallic identifier pins used in this study were less resistant to extreme heat. The significance of these findings is discussed.     

The effect of conditions of putrefaction on species determination in human and animal teeth

Tooth fragments freshly extracted from humans and rats were stored at either 4 degrees C or room temperature in dry or humid conditions for periods ranging from 1 to 6 months. The fragments were reduced to powder and antigens were extracted. Comparison of these samples was carried out using Counter Current Electrophoresis. Extracted sera were tested against known specific antisera and resultant precipitin reactions stained for examination. Correct species identification was possible both from desiccated and humid fragments but there was species variation in the sensitivity of the method. All the extracts from human teeth were positive against human antisera. In the rat some test specimens were initially negative but became positive following further dilution of the extracts.     

The degree of destruction of human bodies in relation to the duration of the fire

The changes occurring during cremation were watched and documented in 15 undissected bodies to be cremated. It was found that at temperatures between 670 and 810°C the body showed the “pugilistic attitude” after about 10 minutes. After 20 minutes the calvaria was free from any soft tissue and fissures of the tabula externa could be noticed. The body cavities became visible after approximately 30 minutes, so that the organs were exposed. Forty minutes after cremation had started, the internal organs were severely shrunken and showed a net-like or sponge-like structure. After about 50 minutes the extremities were destroyed to an extent leaving only the torso which broke apart after 1–1.5 hours. The complete incineration of a human body took about 2–3 hours.     

肋软骨及牙髓细胞DNA含量与PMI的相关性

目的探讨人死后肋软骨及牙髓细胞平均DNA含量与死亡时间(PMI)的相关性,寻求推断PMI的新方法。方法应用细胞图像分析技术分析高温(30～35℃)及低温(15～20℃)环境下人死后0～15d两种组织细胞平均DNA含量的降解情况。结果两种组织细胞平均DNA含量随死后时间的延长而逐步降解,其中低温组牙髓细胞平均DNA含量的降解在死后0～4d内有一个降解平台期。统计分析两组温度下两种组织细胞平均DNA含量与PMI有显著的负相关性(P<0.01)。结论依据两种组织细胞平均DNA含量的降解可进行PMI推断。     

上颌前牙的性别判别分析

根据牙的形态变化判定性别。收集辽宁地区成人上颌牙的石膏模型200例(男、女各100例,年龄在18岁～24岁),利用自动显示游标卡尺测量牙冠厚和牙冠宽,并应用判别分析方法进行分析。得出的6个性别判别式,其判别率为57.5%～64.5%,为用牙的形态变化判定性别提供了依据。     

Univariate sex dimorphism in the Nepalese dentition and the use of discriminant functions in gender assessment

Sex dimorphism in the Nepalese dentition is described using univariate and discriminant analyses. Canines showed the greatest univariate sex dimorphism, followed by the buccolingual (BL) dimension of maxillary first and second molars. Overall, the maxillary teeth and BL dimensions showed greater univariate sex differences. However, less than half of the measured variables (46.4%) showed statistically significant differences between the sexes and the magnitude of sex dimorphism was reduced when compared to other populations. Moreover, reverse dimorphism--where females showed larger teeth than males--was observed in the mesiodistal dimension of mandibular second premolars. This reflects reduction in sexual dimorphism observed through human evolution and the consequent overlap of tooth dimensions in modern males and females. A specific purpose of the study was to develop discriminant functions to facilitate sex classification. A group of functions were developed considering the possibility of missing teeth and/or jaws in forensic scenarios. The functions permitted moderate to high classification accuracy in sexing (67.9% using maxillary posterior teeth; 92.5% using teeth from both jaws). The superior expression of sex dimorphism by means of discriminant functions is in contrast to the univariate results. This is due to discriminant analysis utilising the inter-relationship between all teeth within a dentition--these tooth correlations are not utilised in univariate analysis which results in a loss of information. It is inferred that large-scale statistically significant univariate differences are not a prerequisite for sex assessment.     

Effect of Heat on the Fluorescence Properties of Tooth‐Colored Restorative Materials and Their Forensic Implications

During antemortem and postmortem comparison of dental records of carbonized victims, restorations may be part of such records. The objective of this study was to evaluate the effect of heat on the fluorescence behavior of contemporary tooth‐colored restorative materials and natural tooth structure when subjected to range of temperatures, using illumination with 405 nm wavelength light. A total of 132 human extracted teeth restored with tooth‐colored restorative materials were exposed to heat (200, 500, 900, 1200°C) in an oven for 30 min. Samples were imaged before and after heat treatment. All tooth‐colored restorative materials underwent changes in color and in fluorescence properties, at each of the temperatures used. Resin‐based restorative materials still fluoresced at 200°C, and at 500°C underwent major color changes due to volatilization of resin. Materials containing inorganic fluorophores still fluoresced at 900°C, while at 1200°C, none of the materials tested in this study showed any fluorescence.     

Methods for the analysis of hard dental tissues exposed to high temperatures

The preservation of teeth that have been exposed to a heat source is a key factor in order to keep their value as evidence. This research paper presents some methods under development for the analysis of dental evidence after exposure to high temperatures; it also establishes age related alterations of young and aged teeth which have been exposed to direct heat. A total of thirty permanent healthy teeth (young=15 and old=15) were submitted to direct heat (n=14) and to a controlled raise of temperature (n=16); they were embedded in polymethylmetacrilate. For the macroscopic study, blocks were cut in half, polished and their surfaces were digitalized. Subsequently, blocks were put in slides, worn away and observed through a photomicroscope. The study of the colorimetric pattern was done by Corel Photo Paint 12.0 and Image J. software. All teeth submitted to the gradual raise of temperature presented a degree of structural damage lower than those exposed to direct heat. The internal surface of young teeth exposed to direct heat showed lines of fracture oblique to the long axis of the tooth; in contrast, a reticular pattern was observed on aged teeth. All teeth exposed to direct heat showed some matte-black coloration areas, while those submitted to a gradual increment of temperature showed a greyish colour in the enamel and a chalky-white in the cementum. By using both thermal treatments in young teeth, the amelodentinal junction was lost, thus preserving the integrity of each tissue. In aged teeth, the separation of this junction was produced because of the breakdown of the dentine. Microscopically, in teeth exposed to direct heat, it was neither possible to differentiate the dentin from the cementum, nor to distinguish the incremental lines of the latter; while in those submitted to a controlled increment of temperature, the corresponding morphology in the tissues was observed. A significant statistical difference was obtained for black colour among young and aged teeth exposed to direct heat, as well as among the means of all colours obtained from the teeth exposed to both treatments. The structural changes observed allow locating a tooth in the studied age groups. Our results show the viability of the applied method for the processing of samples, allowing their preservation as evidence during an indefinite time.