Sex determination by PCR analysis of DNA extracted from incinerated, deciduous teeth.

Establishing the biological sex of human remains is a very important part of identifying victims of fire when severe soft tissue destruction has occurred. Deciduous (children's) teeth were exposed to a range of incineration temperatures 100-500 degrees C for 15 minutes. Polymerase Chain Reaction (PCR) amplification was used to identify specific human amelogenin regions. There was successful identification of human biological sex, from deciduous teeth exposed to incineration temperatures of 200 degrees C and below, using standard ethidium bromide gel staining. There was greater sensitivity using fragment analysis by laser induced fluorescence which achieved sex identification from some teeth heated to 400 degrees C.     

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.     

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.     

Rates of putrefaction of dental pulp in the Northwest coast environment

Cytological stability is of interest to criminal investigators in instances where remnants of soft tissue have been preserved, since such tissue can aid in the identification of human remains, helping to determine either the sex of the individual or his or her identity. This study based on seven experiments shows that, in Northwest coast outdoor environments in both summer (three experiments) and winter (three experiments), the stability of dental pulp nuclei ranges from 4 days to 2 weeks. The seventh experiment serves to describe the morphological sequence observed in nuclear putrefaction. The specimens included human and pig extracted teeth and unextracted pig teeth. Deposition of the specimens was made both on the surface and in the subsurface (30-cm depth), and the environmental variables were recorded.     

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).     

Comparative Analysis of the Effects of Heat on the PCR‐Amplification of Various Sized DNA Fragments Extracted from Sus Scrofa Molars*

Abstract: This study examined the effects of heat on the amplification of DNA from the dental pulp of Sus scrofa molars and investigated the protection afforded to the pulp tissue by the dental enamel, alveolar process, and soft tissue of the head. Segments of defleshed maxilla and mandible encasing the first molar (n = 60) were subject to a range of temperatures for 15 min. Dental pulps were retrieved. Amplifications using three‐primer and four‐primer multiplexes showed no degradation of the largest fragment following exposure to 450°C. Amplifications in the three‐primer multiplex (283 bp) were successful following exposure to 525°C in maxillary samples only. This study revealed the enamel density of maxillary molars to be greater than mandibular molars in Sus scrofa. Following incineration of intact heads for 15 min (n = 10) and 1 h (n = 4) at an average temperature of 625°C, amplifications of the largest fragment (450 bp) were successful from both maxillary and mandibular teeth.     

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。     

Color change in dental tissue as a sign of thermal damage

The appearance of hard human tissue after thermal damage permits certain conclusions to be drawn with regard to fire parameters. In addition to morphological destruction, the change in color of dental tissues like enamel, dentine, and cement is also important. We studied teeth extracted from 330 human males and females under well-defined time and temperature conditions. Using the DIN and RAL color indexes, the color phenomenon was evaluated on the basis of the amount of glowing obtained when the teeth were heated. It is easiest to determine the color change in cement, as no calculus or hindering plaques are on the root surface. In addition to this, destruction of the tooth root takes place only during extreme combustion conditions. Determining the dentine color is more difficult and is possible only after the enamel splits. Also, the different dentine thicknesses hinders the observation of color. When the enamel was tested, it was found that the differences in color caused by high temperatures are unimportant. Moreover, the enamel burst into small particles. The effect we observed regarding the anthracite lustre using low temperatures was typical, and the best results were obtained in dental roots with eight color scales. All three dental hard tissues have in common that the variations in color appear regularly and successively according to ascending temperature or duration of time: natural dental color, black, brown, blue, grey, white, and pink. In these processes, the temperature and combustion time are inversely proportional to the velocity of color change. The literature is discussed that deals with temperature- and time-dependent color phenomena of dental hard tissues destroyed as a result of thermal damage.     

Postmortem pink teeth.

A series of cases is reported in which pink teeth were observed during the postmortem period. Most cases were associated with decomposition in a moist environment. Experimental procedures led to the extraction of pink material from dentin and demonstration that hemoglobin and serum proteins were present. The pink-tooth phenomenon was duplicated in human teeth by instilling into the pulp chambers whole blood and blood with the red cells hemolyzed. The change was manifested in teeth of dogs after freezing, heating, and decomposition in a moist environment. The authors postulate that pink teeth occur as a result of breakdown of red blood cells in the pulp chamber of the tooth and diffusion of hemoglobin and other serum proteins into the dentin via the dential tubules. Histochemical studies show that the brown or gray material in some teeth subjected to postmortem aging is probably hemoglobin and serum proteins. Factors of age, vascularity of the pulp chamber, and postmortem conditions are discussed in relation to the postmortem development of pink teeth.     

Long-term storage of blood samples as whole blood at extremely low temperatures for methemoglobin determination

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood and a hemolysate at refrigerated or various freezing temperatures were examined using experimentally prepared blood samples. When whole blood was stored at 3 degrees C, rapid reduction of Met-Hb was observed in the nitrite-treated blood whereas neither reduction nor formation of Met-Hb was observed in the untreated and heated blood within 7 days. When hemolysate was stored at 3 degrees C, Met-Hb concentrations were stable within a few days regardless of the initial values. However, slight autoxidation was observed 7 days after storage in the untreated and heated blood. When whole blood was stored at various freezing temperatures, Met-Hb concentrations were practically stable until at least 30 days at -80 degrees C or -196 degrees C regardless of the initial values, although considerable autoxidation was observed at -30 degrees C especially in the blood containing small amounts of Met-Hb. Based on the results obtained, a new method was devised for long-term storage of whole blood at extremely low temperatures for Met-Hb determinations.     

Degradation of biomolecules in artificially and naturally aged teeth: implications for age estimation based on aspartic acid racemization and DNA analysis

Postmortem teeth are the most stable structures, and can be used to gain different information (age estimation, genetic data). Over long postmortem intervals (PMI), degradation processes may alter the molecular integrity and thus affect the reliability of applied molecular methods. Whereas some knowledge on the degradation of biomolecules in bone during the PMI exists, data for teeth are lacking. In particular, the impact of degradation processes in dentine on age estimation based on aspartic acid racemization (AAR) cannot be estimated yet. Hence, the molecular stability of both collagen and DNA was analyzed systematically, and their impact on the reliability of age estimation based on AAR and genetic analyses was checked. Two hundred and ten human and 59 porcine teeth were heated (90 degrees C in water) to simulate collagen and DNA diagenesis; 14 naturally aged teeth (PMI: 3 days to 1700 years) were analyzed comparatively. Peptide patterns of cyanogen bromide (CNBr)-cleaved collagen were employed as a new approach to check the collagen integrity. In the same samples, collagen yields, amino acid compositions, AAR in different protein fractions, and DNA integrity were analyzed. In heated human and porcine teeth the collagen content declined during the heating experiment. The amino acid composition in human samples was collagen-like until 12 days of heating. In naturally aged teeth, the collagen yielded from 9.5 to 15%, and no discrepancy of amino acid composition to that of modern collagen was observed. Electrophoresis of CNBr-peptides showed an altered pattern in experimentally degraded samples from day 10 on; naturally aged collagen displayed the typical collagen pattern. AAR increased in all protein fractions with increasing duration of the heating experiment; naturally aged samples displayed a slow accumulation of AAR. DNA degraded progressively, and after 32 h of heat exposure no more DNA was detectable, whereas the amplification of nuclear and mitochondrial DNA was successful up to 48 h. STR typing was reliable up to 16 h, and sex determination up to 40 h of heat exposure. In naturally aged samples of DNA quality, yield and typing success did not correlate with PMI. The data highlight a remarkable stability of collagen dental proteins. Within relevant forensic periods a postmortem rise of AAR under normal conditions is negligible, and analyses of dental DNA has a high chance to be successful. However, after large PMI and/or extreme postmortem conditions age estimation based on AAR and genetic analyses lose their reliability.     

Development and validation of a liquid chromatography-mass spectrometry assay for hair analysis of methylphenidate

Examination of various SIM cards and smart card devices indicates that data may be retained in SIM card memory structures even after heating to temperatures up to 450 degrees C, which the National Institute of Standards and Technology (NIST) has determined to be approximately the maximum average sustained temperature at desk height in a house fire. However, in many cases, and certainly for temperatures greater than 450 degrees C, the SIM card chip has suffered structural or mechanical damage that renders simple probing or rewiring ineffective. Nevertheless, this has not necessarily affected the data, which is stored as charge in floating gates, and alternative methods for directly accessing the stored charge may be applicable.     

AmpFlSTR Profiler Plus and AmpFlSTR COfiler analysis of tissues stored in GenoFix, a new tissue preservation solution for mass disaster DNA identification

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.     

Detection of methamphetamine in mouse femurs exposed to high temperature

Bone samples are valuable for examining the cause of death and circumstance leading up to death when body fluids are not available for forensic toxicological analysis. Examined were heat-induced changes in methamphetamine and amphetamine concentrations in femurs removed from methamphetamine-injected mice to determine if the burned bones could be used for toxicology testing. The femurs were heated at 100°C, 300°C, or 500°C for 10 or 30 min. The tissue structure of the heated femurs was preserved at 100°C for 30 min but was destructed at higher temperatures. Methamphetamine and amphetamine were detected in femurs heated at 100°C for 10 min, 100°C for 30 min, and 300°C for 10 min (with methamphetamine and amphetamine concentrations ranging from 0.36 to 35 μg/g and 0.54 to 47 μg/g, respectively). Methamphetamine and amphetamine were detectable when heated above their decomposition temperature as a result of limited heat transfer do to protection provide by the femoral muscle. Thus, the bone could be a useful analytical sample in cases of burn-related deaths, where it is difficult to collect body fluids.     

Analysis of a soldered wire burnt in a fire

Fire investigators frequently encounter electrical wires with melted ends at fire scenes, which can provide useful information on the cause and development of the fire even when the melted ends result from the fires. A bead on a melted end of a wire was found in the area of origin of a massive fire that lasted for nearly a whole day, devastating a factory. The bead appeared to be the end of a wire soldered to a terminal. X-ray analysis showed that the bead is a tin-copper alloy. Although regular tin-lead solder was used in the factory, lead was not detected. This contradictory result stood in the way of fire investigation. Therefore, an unused wire soldered with tin-lead solder was heated in an electric furnace at 500 degrees C for 3 h for further examination. X-ray analysis of this wire showed that copper can be alloyed with tin while losing lead in a long-term heat in a fire.     

Phosphoglucomutase isoenzymes in human teeth

Powdered dentine samples were prepared from fresh teeth and teeth allowed to putrefy for up to 90 days. In some samples dentine only was included, whereas in others dentine plus pulp remnants were used. Extracts of the powders were tested for phosphoglucomutase activity by starch gel electrophoresis and PGM typing was carried out. Fresh teeth showed activity in at least 50% of cases as did putrefied dentine plus pulp remnants. Putrefied dentine alone showed minimal activity. The method may be useful for identification procedures using fragments of teeth.     

alpha-L-fucosidase phenotyping in human tissues, dental pulps and hair roots

Phenotypes of alpha-L-fucosidase (Fu) were demonstrated from tissue samples of spleen, liver, lung and kidney stored for a few weeks at room temperature. Activity was fairly low in pancreas, heart, muscle, skin and brain, and typing was not reliable after 1 week storage. Fu types were detectable from dental pulp tissue of up to 1 week storage. Activity was present in hair root cells, but was extremely low. The results show that the Fu typing may be applicable to individual identification of organ tissues and teeth.     

Comparison of gasolines using gas chromatography-mass spectrometry and target ion response

Gas chromatography-mass spectrometry was used to compare gasoline samples obtained from different sources based on the difference in amounts of certain components found in the headspace of gasoline using target response data. Many suspected arson cases involve comparing an ignitable liquid extracted from fire debris to a liquid found in a suspect's possession to determine if they could have had a common source. Various component ratios are proposed for determining if an unevaporated gasoline sample could have originated from the same source as an evaporated gasoline extracted from fire debris. Fifty and 75% evaporated gasoline samples were both found to contain similar ratios of certain components when compared with its unevaporated source gasoline. The results of the comparisons in this study demonstrate that for cases involving gasoline that has been evaporated up to 50% and extracted from pine, it is possible to eliminate comparison samples as originating from the same source. The results of the 75% comparisons suggest it may be possible to apply the same type of comparison to cases involving 75% evaporated gasoline.     

Flow cytometric evaluation of postmortem pulp DNA degradation

During postmortem autolysis, cellular organelles and nuclear DNA break down into their constituent parts. DNA flow cytometric analysis was applied to study the denaturation of splenic cell DNA as a possible method for postmortem interval determination. DNA denaturation continued for 72 hours at a constant rate, with no intact DNA peaks thereafter. The value of using dental pulp tissue for flow cytometric determination of postmortem interval was investigated. The pulps of 57 routinely removed impacted third molars from patients 15 to 30 years of age were obtained. Pulp tissue was removed at 24, 48, 72, 96, 120, and 144 hours postextraction. Debris (degraded DNA) was defined as all signals left of the standardized mean 2n peak and expressed as a percentage of the total number of signals. In contrast to the splenic cell DNA, dental pulp tissue exhibited minimal DNA degradation by 144 hours postextraction, and no constant relation was found between time and DNA degradation during this time. In this study, pulp tissue was found to be unreliable to determine the early postmortem interval but might be of greater value in the later stages.     

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.