Dating human skeletal remains using a radiometric method: biogenic versus diagenetic 90Sr and 210Pb in vertebrae

In forensic science, there is a strong interest in determining the post-mortem interval (PMI) of human skeletal remains up to 50 years after death. Currently, there are no reliable methods to resolve PMI, the determination of which relies almost exclusively on the experience of the investigating expert. Here we measured (90)Sr and (210)Pb ((210)Po) incorporated into bones through a biogenic process as indicators of the time elapsed since death. We hypothesised that the activity of radionuclides incorporated into trabecular bone will more accurately match the activity in the environment and the food chain at the time of death than the activity in cortical bone because of a higher remodelling rate. We found that determining (90)Sr can yield reliable PMI estimates as long as a calibration curve exists for (90)Sr covering the studied area and the last 50 years. We also found that adding the activity of (210)Po, a proxy for naturally occurring (210)Pb incorporated through ingestion, to the (90)Sr dating increases the reliability of the PMI value. Our results also show that trabecular bone is subject to both (90)Sr and (210)Po diagenesis. Accordingly, we used a solubility profile method to determine the biogenic radionuclide only, and we are proposing a new method of bone decontamination to be used prior to (90)Sr and (210)Pb dating.     

An estimation of the post-mortem interval in human skeletal remains: a radionuclide and trace element approach

Estimation of the post-mortem interval remains a contentious issue, with forensic pathologists often relying upon the recognition of morphological changes.A radionuclide approach has often been suggested in the literature, although limitations have prevented its application, most notably those of diagenesis. Within this pilot study, we show for the first time that there is a correlation between certain radionuclide content and time since death.A larger study is proposed to confirm these findings and possibly provide a calibration against which bones uncovered can be dated.     

The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth

Estimation of age-at-death for skeletonised forensic remains is one of the most significant problems in forensic anthropology. The majority of existing morphological and histological techniques are highly inaccurate, and show a bias towards underestimating the age of older individuals. One technique which has been successful in forensic age estimation is amino acid racemization in dentine. However, this method cannot be used on remains where the post-mortem interval is greater than 20 years. An alternative approach is to measure amino acid racemization in dental enamel, which is believed to be more resistant to change post-mortem. The extent of amino acid racemization in the acid soluble fraction of the enamel proteins was determined for modern known age teeth. A strong correlation was observed between the age of the tooth and the extent of racemization. No systematic bias in the direction of age estimation errors was detected. For the majority of teeth analyzed, the presence of dental caries did not affect the results obtained. In a minority of cases, carious teeth showed a higher level of racemization than would be expected given the age of the individual. These results indicate that amino acid racemization in enamel has the potential to be used in age estimation of skeletal remains.     

Post-mortem changes in calmodulin binding proteins in muscle and lung

Estimation of post-mortem interval (PMI) remains an elusive issue in forensic investigations. In this study, we examined the possible use of calmodulin (CaM) binding proteins (CaMBPs) as indicators of PMI. Whole CaMBP populations from homogenized rat lung and rat skeletal muscle removed at 0, 24, 48 and 96 h post-mortem at 21 degrees C were detected by the calmodulin binding overlay technique (CaMBOT) using 35S-VU1-CaM and visualized by autoradiography. CaMBOT showed that, in both tissues, the CaMBP population remained relatively stable for up to 96 h post-mortem with the exception of a single approximately 200 kDa CaMBP that increased in 24 h post-mortem samples then showed decreasing amounts at subsequent times. Immunoblot analysis of the specific CaMBPs, Ca(2+)/CaM-dependent kinase II (CaMKII), calcineurin A (CNA), myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (iNOS) were done on lung tissue samples. CaMKII levels did not change appreciably over the 96 h PMI examined. In contrast to iNOS levels, which varied from sample to sample, CNA and MARCKS showed predictable patterns of change: the level of MARCKS decreased steadily in the 0-96 h post-mortem lung samples while CNA underwent a shift in mobility on SDS-PAGE by 24 h post-mortem before slowly decreasing in amount. The stability of CaMKII levels over 96 h was also seen in skeletal muscle tissue while CNA showed variable levels at 0, 48 and 96 h with the presence of the rapidly migrating band at 24 h. These patterns of change in CaMBPs provide some insight into the post-mortem changes in calmodulin-mediated signaling components in lung and skeletal muscle and support the further study of CNA and CaMKII as potential markers for estimating short- and long-term PMIs.     

Blood carbon monoxide and hydrogen cyanide concentrations in the fatalities of fire and non-fire associated civil aviation accidents, 1991-1998

To evaluate pathophysiological significance of post-mortem urinary myoglobin levels in determining the cause of death, we investigated 210 forensic autopsy cases, partially in comparison with serum levels. Post-mortem serum myoglobin levels were extraordinary high in most cases possibly due to post-mortem change. Urinary myoglobin levels did not correlate with the serum levels, showing possible post-mortem elevation in cases of a prolonged post-mortem period over 48h. A high (>1000 ng/ml), moderate (100-1000 ng/ml), slight (50-100 ng/ml) and not significant (<50 ng/ml) elevation of urinary myoglobin were observed in 26, 43, 31 and 110 cases, respectively. Half the highly elevated cases were those with a survival time over 24h. In cases of minor muscle injury such as head trauma, elevation of urinary myoglobin level was closely related to longer survival. In acute/subacute deaths with a post-mortem interval within 48h, a significant difference was observed in relation to the blood carboxyhemoglobin (COHb) levels of fire victims: myoglobinuria over 100 ng/ml was more frequently and markedly observed in cases with COHb below 60% than over 60%, suggesting muscle damage in fatal burns. Similar elevation was observed in heat stroke victims, and also in some cases of acute and subacute death from polytrauma, asphyxiation, drowning, electricity and spontaneous cerebral bleeding, but not in myocardial infarction. Thus, it was suggested that high post-mortem urinary myoglobin levels in acute and subacute death cases may be a possible indicator of antemortem massive skeletal muscle damage as well as exertional muscle hyperactivity or convulsive disorders associated with hypoxia.     

Differences in morphological age-related dental changes depending on postmortem interval.

Morphological methods for age estimation from teeth have been developed and applied to samples without taking the postmortem interval into consideration. We studied differences in morphological age-related changes between fresh extracted teeth and teeth from human skeletal remains in order to develop appropriate dental age estimation methods according to the time after death. Forty-three permanent teeth from dental patients were compared to 37 teeth obtained from human skeletal remains with a postmortem interval from 21 to 37 years. Morphological age-related changes were investigated by measuring variables on intact and half-sectioned teeth. A new computer assisted image analysis procedure to avoid subjectivity was developed to measure variables in sectioned specimens. Dental color, translucency length, attrition, cementum apposition, and secondary dentin showed higher values in teeth from human skeletal remains than in fresh extracted teeth. Variables obtained by morphometric analysis of computer-generated images (tooth length, tooth width, root length, and root area) showed higher values in fresh extracted teeth than in teeth from skeletal remains. The postmortem interval affects age-related morphological changes, and therefore different methods should be used for teeth of unknown postmortem interval.     

Tetracycline-like fluorescence in buried human skeleton parts

Some ubiquitous soil-living micro-organisms are producing tetracycline-like fluorescences in skeletal remains. For this reason, the possibility of using tetracycline fluorescences in dating or allocating displaced skeletal parts is severely limited.     

A New Method for Determination of Postmortem Interval: Citrate Content of Bone*

Abstract: Few accurate methods exist currently to determine the time since death (postmortem interval, PMI) of skeletonized human remains found at crime scenes. Citrate is present as a constituent of living human and animal cortical bone at very uniform initial concentration (2.0 ± 0.1 wt %). In skeletal remains found in open landscape settings (whether buried or not), the concentration of citrate remains constant for a period of about 4 weeks, after which it decreases linearly as a function of log(time). The upper limit of the dating range is about 100 years. The precision of determination decreases slightly with age. The rate of decrease appears to be independent of temperature or rainfall but drops to zero for storage temperature <0°C.     

Estimating the pre-appearance interval from temperature in Necrodes littoralis L. (Coleoptera: Silphidae)

While estimating post-mortem interval from entomological evidence it is useful to divide it into two separate intervals: the pre-appearance interval (PAI) and the development interval in the case of immature insects and PAI and the presence interval in the case of adult insects. An accurate estimate of PAI is critically important particularly in the case of those insects which appear on remains late in decomposition. In this paper the possibilities of PAI estimation from temperature are evaluated in late-arriving, forensically important carrion beetle Necrodes littoralis. The relationship between PAI of adult and larval N. littoralis and temperature was modelled by using results of succession experiments in forests of Central Europe. In both stages PAI was found to be strongly, inversely dependent on mean daily ground level temperatures averaged for the duration of PAI. Several methods of PAI estimation were proposed and their accuracy was initially evaluated with external successional data.     

Postmortem interval of skeletal remains through the detection of intraosseal hemin traces. A comparison of UV-fluorescence, luminol, Hexagon-OBTI®, and Combur® tests

With the goal of obtaining additional practically applicable methods for estimating the PMI of skeletal remains, 39 samples of human and 5 samples of domestic animal long bones with known PMI (PMI=1 to approximately 2000 years) were tested with two established methods (UV-fluorescence of a freshly sawn cross-section and the luminol test) and two screening tests (Hexagon-OBTI? test and Combur? test) that were being tried out in this context for the first time. The hypothesis underlying this experiment was the supposition that the PMI-related chemiluminescence of the luminol reaction for bone is based on the presence of persisting hemin from hemoglobin molecules in bone. Our results showed that lack of luminescence and reduced UV-fluorescence were more meaningful results for estimating PMI and excluding forensic relevance than a positive luminol reaction or strong UV-fluorescence, as both of the latter findings revealed the limitations of these methods in this particular context. Particularly for cases showing a positive luminol reaction, the use of additional absolute dating methods may be indicated. Against our expectations, both the Combur? test strips and the Hexagon-OBTI? test, which were both devised to demonstrate blood, delivered negative results for all samples. They are thus not suitable for estimating the PMI of skeletal remains. Future research will be necessary to elucidate whether the negative results obtained for these tests may be due to the poor solubility of potentially present hemoglobin or hemoglobin breakdown products in the Tris buffer used in this experiment.     

Gender Determination of Adult Individuals by Three‐Dimensional Modeling of Canines

Gender determination is a fundamental issue in forensic anthropology. Many techniques based on bone and dental remains have been proposed. It is not always possible to implement the techniques using bones, but teeth are often perfectly preserved. It has been demonstrated that the canine has the greatest sexual dimorphism, and the aim of this work was to provide an easy and accurate dental technique for determining the gender in the absence of other skeletal elements. The sample was composed of 210 CT scans with four healthy canines. The 840 canines were modeled using MIMICS^® 10.01 software. The total volume of each tooth was determined. Seven mathematical models were determined by binary logistic regressions and ranked in order of relative performance. The seven proposed predictive models thus performed (0.910 ≤ AUC ≤ 0.938), with overall rates of correct predictions between 82.38 and 85.24%. The 4‐canine model is the most powerful for predicting the gender.     

Accuracy of developing tooth length as an estimate of age in human skeletal remains: the deciduous dentition

Dental age assessments are widely used to estimate age of immature skeletal remains. Most methods have relied on fractional stages of tooth emergence and formation, particularly of the permanent dentition, for predicting the age of infants and very young children. In this study, the accuracy of regression equations of developing deciduous tooth length for age estimation (Liversidge et al.) is tested on a sample of 30 Portuguese subadult skeletons of known age at death. Overall the method shows high accuracy and the average difference between estimated and chronological age is between 0.20 and -0.14 years when using single teeth, and 0.06 years, when using all available teeth. However, there is a tendency for the deciduous molars to provide overestimates of chronological age. Results show that age estimates can be obtained within +/-0.10 years with a 95% confidence interval when several teeth are used. Overall between-tooth agreement in age estimates decreases with increasing age but there is less variability of estimates with more teeth contributing to overall mean age. One seemingly limitation of this method may be the fact that it was developed by combining the maxillary and mandibular teeth. The other is related to the accuracy with which radiographic tooth length can be used as a valid surrogate for actual tooth length. Nevertheless, the advantages of this metric method surpass the limitations of chronologies based on stages of dental development.     

A Review of Bomb Pulse Dating and its Use in the Investigation of Unidentified Human Remains

In cases where there is limited antemortem information, the examination of unidentified human remains as part of the investigation of long-term missing person's cases is a complex endeavor and consequently requires a multidisciplinary approach. Bomb pulse dating, which involves the analysis and interpretation of 14C concentration, is one technique that may assist in these investigations by providing an estimate of year of birth and year of death. This review examines the technique of bomb pulse dating and its use in the identification of differentially preserved unknown human remains. Research and case studies implementing bomb pulse dating have predominantly been undertaken in the Northern Hemisphere and have demonstrated reliable and accurate results. Limitations were, however, identified throughout the literature. These included the small sample sizes used in previous research/case studies which impacted on the statistical significance of the findings, as well as technique-specific issues. Such limitations highlight the need for future research.     

Application of the hexagon-OBTI test and the RSID blood test for the determination of the post-mortem interval of bone samples

In this serial experiment, five human bones with known post-mortem intervals (PMI) in a soil environment from five different epochs (0.2 to approximately 2000 years) were tested in a blind setup with two established rapid tests for the identification of human blood traces (Hexagon-OBTI test and RSID blood test). Based on previous study results concerning the usability of the Luminol test for the first assessment of the PMI of osseous remains, the question arising was whether those test procedures, which are highly sensitive for the detection of human blood components, could also be used to narrow down the post-mortem interval. Five test series were conducted applying modified standard protocols of the manufactures. The aim was to find out whether with prior reaction steps or a prolonged time of incubation hemoglobin or its metabolites can be dissolved from the bone and positive test results can be achieved dependent on the PMI. Four test series yielded negative results for all bone samples and one test series a uniformly weak positive result. The results indicate that rapid tests based on the detection of blood are not suitable for the determination of the PMI of bone samples despite the modification of the standard protocols. Further thorough research is required to clarify the postmortem degradation of hemoglobin in bones.     

Different skeletal elements as a source of DNA for genetic identification of Second World War victims

To this day process of identification of missing persons from skeletonized human remains with help of forensic genetics proves to be complex and challenging. The success rate of genetic identification in bones strongly depends on a combination of various factors, most importantly environmental factors and post-mortem interval. Furthermore, there are individual-specific factors that affect DNA preservation, such as race, gender, age and type of skeletal elements. The goal of our study was to optimize sampling process through determining which skeletal elements are superior in their preservation of DNA in 70-yearold skeletons belonging to victims of Second World War. We sampled different types of bones and teeth from three such skeletons found in Slovenian hidden mass grave Huda jama, 56 elements from each respective skeleton, together 168 elements. With the help of parameters, such as quantity of DNA, degradation rate and typing success, we tried to find the best types of elements to identify the victims. Prior to powdering bones and teeth, we removed contaminants. We decalcified 0.5 g bone and tooth powder followed by extraction and purification of DNA using Biorobot EZ1 (Qiagen). Quantification of obtained nuclear DNA was carried out using PowerQuant kit (Promega) and autosomal STR typing using ESSplex SE QS kit (Qiagen). Best parameters to assess skeletal elements that are superior in their DNA preservation were quantity of DNA and number of successfully typed STR loci. Metacarpal and metatarsal bones proved to be the best, followed by intermediate cuneiform, first distal foot phalanx, talus, petrous bone and tibia. We also created elimination database for persons involved in exhumation, anthropological and genetic analyses and exclude potential contamination.     

Radiocarbon dating as tool to assist in triaging cases of unidentified human remains in Victoria,Australia: A case series

In cases where human remains are unidentified because there is no initial identification hypothesis, limited contextual information, and/or poor preservation, radiocarbon (¹⁴C) dating may be a useful tool to further assist with identification. Through measuring the amount of ¹⁴C remaining in organic material, such as bone, teeth, nail, or hair, radiocarbon dating may provide an estimated year of birth and year of death for a deceased person. This information, may assist in, establishing whether a case of unidentified human remains (UHR) is actually of medicolegal significance and therefore, requires forensic investigation and identification. This case series highlights the application of ¹⁴C dating to seven of the 132 UHR cases in Victoria, Australia. Cortical bone was sampled from each case and the level of ¹⁴C was measured to provide an estimated year of death. Four of the seven cases analyzed contained the levels of ¹⁴C consistent with an archeological timeframe, one contained a level of ¹⁴C consistent with a modern (i.e., of medicolegal significance) timeframe, and the results for the remaining two samples were inconclusive. Applying this technique not only reduced the number of UHR cases in Victoria but also has investigative, cultural, and practical implications for medicolegal casework in general.     

Multiple regression models for age estimation by assessment of morphologic dental changes according to teeth source

The aims of this study were twofold: (1). to measure parameters that contribute significantly to estimates of dental age, using a combination of classic methods and a computer-assisted image analysis procedure to avoid the bias inherent in observer subjectivity; and (2). to develop new mathematical regression models for age prediction according to postmortem interval. Two different populations were studied. Forty-three permanent teeth (Group I), extracted for valid clinical reasons, were taken from patients 25-79 years of age. The other population group (Group II) was composed of 37 healthy erupted permanent teeth obtained from human skeletal remains (age 22-82 years) with a postmortem interval ranging from 21 to 37 years. Morphologic age-related changes were investigated by measuring variables on intact and half-sectioned teeth. Multiple regression analyses were performed with age as the dependent variable for each sample source. In fresh extracted teeth, the variables that made the greatest contributions to predictions of age were dental attrition, dentin color, and translucency width, the latter measured with a computer-assisted image analysis method. In teeth from human skeletal remains, the variables that made the greatest contributions to age calculation were cementum apposition, pulp length measured by computer-assisted image analysis, dental attrition, root translucency, and dental color. We conclude by recommending different regression models to calculate age depending on the postmortem interval.     

Practice of forensic entomology--usability of insect fragments in the estimation of the time of death

Forensic entomology analyzes and evaluates the presence of necrophagous or necrophilic insects on human corpses to estimate the post-mortem interval. The three cases presented are good examples illustrating that not only the various stages of development of the necrophagous insects from egg via larva to pupa are useful for forensic assessment. Depending on the situation in the individual case, even inconspicuous remains of past insect activity such as empty pupal cases or larval skins may supply important entomological information. For this purpose it is indispensable to preserve such evidence, which may appear only as fragments or pieces at first sight, for proper entomological assessment.     

A preliminary study of changes in scalp impedance during the early post-mortem period in rats.

An accurate and reproducible technique was used for estimation of scalp impedance (1 kHz) in each of eight rat cadavers, maintained at 9.0 +/- 0.7 degrees C, during the 1 to 144 hour post-mortem period. Scalp impedance increased non-linearly, and in a bimodal manner, between 24 and 144 hours post-mortem. The relationship between scalp impedance and post-mortem interval, and the degree of individual variation in the time course of impedance change, were such that scalp impedance is unlikely to be a suitable tool for estimation of time since death.     

骨密度检测在野生动物司法鉴定领域的应用前景

脊椎动物的体内存在着骨骼,骨密度代表了骨骼中骨矿物质的含量.通过对骨密度测量的多种方法及其基本原理进行介绍.因不同种属动物骨骼存在着差异,通过实验验证测量“面密度”的骨密度检测方法在动物骨骼检测时存在着很大的局限性,定量CT法（Qcantitative computed Tomography,QCT）测量的是“体密度”,能够避免各种“面密度”检测方法的缺陷,在动物骨密度检测时应当作为首选.最终提出骨密度检测方法可应用于动物司法鉴定领域.