The Potential of High-throughput Metagenomic Sequencing of Aquatic Bacterial Communities to Estimate the Postmortem Submersion Interval

Human remains can be discovered in freshwater or marine ecosystems, circumstances where insects and other invertebrates have infrequently been used for understanding the time of postmortem submersion. In this study, the identification and succession of epinecrotic bacterial communities on vertebrate remains were described during decomposition in a temperate headwater stream during two seasons (summer and winter). Bacterial communities were characterized with 454 pyrosequencing and analyzed at phyletic and generic taxonomic resolutions. There was a significant increase in genera richness over decomposition during both seasons. Additionally, multivariate statistical modeling revealed significant differences in bacterial communities between seasons at both taxonomic resolutions and siginificant genera differences among sampling days within each season, suggesting a succession of these communities. These data are the first to describe aquatic bacterial succession using high-throughput metagenomic sequencing on vertebrate remains submerged in a freshwater habitat, and provide initial evidence for their potential use in forensic investigations.     

Evaluation of postmortem serum calcium and magnesium levels in relation to the causes of death in forensic autopsy

There appears to be very poor investigation of postmortem serum calcium (Ca) and magnesium (Mg) for diagnostic evidence to determine the cause of death. The aim of the present study was a comprehensive analysis of the serum levels in relation to the causes of death in routine casework. Autopsy cases (total, n=360; 5-48 h postmortem), including blunt injury (n=76), sharp injury (n=29), asphyxiation (n=42), drownings (n=28: freshwater, n=11; saltwater, n=17), fire fatalities (n=79), methamphetamine (MA) poisoning (n=8), delayed death from traumas (n=37), and acute myocardial infarction/ischemia (AMI, n=61), were examined. In total cases, there was no significant postmortem time-dependent rise in serum Ca and Mg. Both Ca and Mg levels in the heart and peripheral blood were significantly higher in saltwater drowning compared with those of the other groups. In addition, a significant elevation in the Ca level was observed in freshwater drowning and fire fatalities, and in the Mg level in fatal MA intoxication and asphyxiation. Topographic analyses suggested a rise in serum Ca and Mg due to aspirated saltwater in drowning, that in serum Ca in freshwater drowning and fire fatalities of peripheral skeletal muscle origin and that in serum Mg in MA fatality and asphyxiation of myocardial and/or peripheral origin. These markers may be useful especially for diagnosis and differentiation of salt- and freshwater drownings and may be also helpful to determine the causes of death involving skeletal muscle damage, including burns and MA intoxication.     

Classifying serial killers

We attempted to match the appearance and demeanor of 27 serial killers to the postmortem 'signatures' found on their victims' bodies. Our results suggest that a link may exist between postmortem signatures and two complementary appearance-demeanor types.     

Pleural effusion in bodies recovered from water

Both in saltwater and freshwater drowning cases, a common autopsy sign is pleural effusion. However, the factors that determine the amount of pleural effusion have not been well established. An attempt was therefore made to correlate the amount of pleural fluid in bodies recovered from water with several parameters registered on the judicial files as well as autopsy findings from the years 1994-1998. The number of cases with pleural fluid increase was found to be very high in saltwater drowning (P<0.001). But, when the freshwater and saltwater drowning cases with pleural fluid increase were compared according to pleural fluid amount, no significant difference was detected (521+/-340 and 768+/-536 ml, respectively). Although there was a positive correlation between the decomposition degree and the fluid in the pleural cavity, a relative decrease was detected in the amount of effusion contrary to the expectations in cases of extreme decomposition. Pleural fluid amount provides significant data about the type of water and the cause of death in early postmortem interval. And there is a link between the time spent in water and the amount of pleural effusion. With the advance of the postmortem interval, decomposition level and the duration of immersion should be taken into account in differential diagnosis.     

Putrefaction: An Additional Complicating Factor in the Assessment of Freshwater Drownings in Rivers

Background hypothesis: That bodies immersed in freshwater (rivers) may show greater changes of decomposition than bodies immersed in saltwater (sea). Fifty de‐identified river drownings and 50 sea drownings were randomly selected from autopsy files at Forensic Science SA, Adelaide, Australia and assessed for decomposition. Results: The river cases consisted of 43 males and seven females (M:F 6:1); age range of 2–92 years (mean 46.8 years); decomposition—16/50 (32%); postmortem interval 1–14 days, average 3.8. The sea cases consisted of 36 males and 14 females (M:F 2.6:1); age range of 9–81 years (mean 50.7 years); decomposition—2/50 (4%) (p < 0.001%); postmortem interval 1–6 days, average 2.8. As decomposition reduces information obtained at autopsy, postmortem examination of deaths in rivers may be less informative than deaths that have occurred in the sea. The reasons for less decomposition in the sea include cooler temperatures, exposure to salt and shorter postmortem intervals.     

蛋白质降解与死亡时间推断的初步研究

目的观察大鼠肝脏肌动蛋白、微管蛋白在死后不同时间的降解情况,为死亡时间的推断寻找客观依据。方法大鼠麻醉致死后置于21℃温度控制系统模拟死后18d的改变,在不同时间点剪取肝脏组织抽提蛋白质,利用westernblot技术检测肌动蛋白、微管蛋白的降解变化并对产物进行半定量分析。结果大鼠肝脏肌动蛋白在死后8d尚可检测,死亡10d后检测不到;在死后2d,α微管蛋白已检测不到,但可检测到β微管蛋白,死亡4d后,β微管蛋白也检测不到。结论肌动蛋白、微管蛋白的死后降解存在差异,其在肝脏组织保存时间的不同可作为死亡时间推断的参数。     

A brief history of forensic entomology.

Apart from an early case report from China (13th century) and later artistic contributions, the first observations on insects and other arthropods as forensic indicators were documented in Germany and France during mass exhumations in the late 1880s by Reinhard and Hofmann, whom we propose recognizing as co-founders of the discipline. After the French publication of Mégnin's popular book on the applied aspects of forensic entomology, the concept quickly spread to Canada and the US. At the time, researchers recognized that the lack of systematic observations of forensically important insects stood in the way of their use as indicators of postmortem interval. General advances in insect taxonomy, and ecology helped close this gap over the following decades.Many early case reports dealt with alleged child homicides, including the suspected use of sulphuric acid. In this context, it was shown that ants, cockroaches, and freshwater arthropods could produce postmortem artifacts suggestive of child abuse.After the World Wars, few forensic entomology cases entered the scientific literature. From the 1960s to the 1980s, Leclecq and Nuorteva were primarily responsible for maintaining the method in Central Europe, with a focus on case work. Since then, basic research in the US, Russia and Canada has opened the way to the routine use of entomology in forensic investigations. The following article gives a brief overview of historic developments in the field. A major focus is on the work done between 1850 and 1950. Since sources from that time remain difficult to track down, the article also includes a historic bibliographical overview on forensic entomology of that era.     

Postmortem Intervals in Mice Submerged in Aqueous Environments at 20°C

Aqueous submersion can impede decay and produce decomposition stages not seen in terrestrial burials. To further understand the variances, fifty-four mice were submerged in freshwater, marine water, and a control environment at 20°C. The mice displayed sequential stages at differing rates over 6 weeks. Regression plots and comparative t-tests demonstrated that internal putrefaction, weight difference, and abdominal circumference of the aqueous environments varied significantly from the control group. The aqueous subjects did not vary significantly from each other quantitatively. The postmortem intervals were not consistent regardless of temperature or environment although a clear variance was noted between the control and the submerged groups.     

Visualization of postmortem chondrocyte damage by vital staining and confocal laser scanning 3D microscopy

The present study was designed to investigate whether the combination of vital dyes [calcein acetomethyl ester and ethidium homodimer (LIVE/DEAD Viability/Cytoxicity Kit)] together with confocal laser scanning 3D microscopy was a suitable process to detect postmortem chondrocyte damage, and whether this process could be used to establish postmortem interval. Human knee cartilage from 13 autopsies (postmortem interval from 1 day to 2.5 months) was incubated with the two dyes. The chondrocytes revealed intense staining according to their vitality. For those cases that were stored mainly at 4 degrees C there was a vitality of approximately 88 to 96% within the first 4.5 days, which decreased to 58% after 6 days and to 9% after 1.5 months. After 2 days and 14 days at summer temperatures there were 70% and 8% vital chondrocytes respectively. Three of the 13 cases showed that altered body and storage conditions limited the efficacy of the method. Initial data suggested a time and temperature dependent increase in cell breakdown. Under stable cooling conditions the use of vital dyes and confocal laser scanning 3D microscopy to measure chondrocyte loss may be a valuable tool for estimating the postmortem interval.     

Postmortem determination of the biological distribution of formic acid in methanol intoxication.

Two 25-year-old men were fatally intoxicated with methanol. The formic acid levels in their blood, urine, and organs were determined postmortem by headspace gas chromatography. The postmortem concentrations of formic acid in the two patients were the following: 0.32 and 0.23 mg/mL in blood, 2.27 and 0.47 mg/mL in urine, 0.11 and 1.17 mg/g in the brain, 0.54 and 0.51 mg/g in the liver, and 0.13 and 1.19 mg/g in the kidneys. The total amounts of formic acid in the gastric contents were 108 and 23.2 mg.     

Hemolytic staining of the intima of the aortic root in freshwater drowning: a retrospective study

A retrospective study was conducted of files at the Institute of Legal Medicine in Hamburg, Germany from 1998 to 2003 of all cases of drowning where there was no putrefaction, to document the rate of recording of hemolytic staining of the intima of the great vessels. All cases had full police investigations with reviews of previous histories, circumstantial evidence, and autopsy findings. A series of control subjects who had died of various natural and nonnatural, nondrowning deaths, matched for age, sex, and postmortem interval, was also reviewed. One hundred twenty cases of freshwater drowning were identified. Drowning occurred in a garden pool in 1 case, in a bath in 2, and in the river Elbe in 117. The age range was from 2 to 91 years (mean = 55 years; M:F = 1:1.8). Hemolytic intimal staining of the aortic root was documented in 6 cases (6 of 120; 5%). This consisted of reddened discoloration of the proximal portion of the aortic root, without any significant staining of the proximal pulmonary artery. No significant hemolytic staining of the intima of the great vessels was recorded in any of the 120 control cases. Although under-reporting of findings may occur in retrospective analyses, this study has shown that at least 5% of freshwater drowning cases showed differential staining of the pulmonary trunk and aorta, with hemolytic discoloration of the aortic intima. When present, hemolytic staining of the aortic root intima may be a useful and possibly under-recognized corroborative sign of freshwater drowning.     

The presence of trace amines in postmortem cerebrospinal fluid in humans

The postmortem levels of biogenic amines in cerebrospinal fluid may represent a useful tool in defining some pathological conditions; no information is available concerning the occurrence of trace amines in postmortem cerebrospinal fluid. Thus, the occurrence of octopamine, synephrine and tyramine were evaluated by using a HPLC system in 20 postmortem samples of cerebrospinal fluid (obtained from 11 males and 9 females) and their levels were compared with those of 20 living subjects (obtained from 11 males and 9 females). The results show that trace amines dramatically increase in the postmortem cerebrospinal fluid (100, 20, and 4 fold increase for tyramine, octopamine, and synephrine respectively). To our knowledge, our data represent the first time trace amines have been identified in postmortem cerebrospinal fluid and the dramatic increase observed for tyramine has the potential of becoming a new tool in forensic science for better defining the time of death.     

Factors affecting postmortem tooth loss

Unassociated human bones are a particular problem during the exhumation of mass graves and a factor that limits anthropological and paleopathological analyses from archaeological contexts. Extensive anthropological literature has focused on the complex taphonomic factors that influences bone assemblages, but little attention has been paid to postmortem tooth loss and factors affecting this process. The following study focuses upon the influence of different factors on postmortem tooth loss. Three samples were investigated in the study: a medieval church cemetery containing 110 individual skeletal remains, and two samples from a series of mass graves made within the same time period in 1999, containing 402 bodies. The frequency of postmortem tooth loss was analyzed relative to postmortem interval for each sample, excavation methods, age distribution, and presence of bone loss associated with periodontal disease. Our results indicate that the degree of alveolar bone loss significantly affected both antemortem and postmortem tooth loss and that the frequency of postmortem tooth loss has the strongest correlation to time since death. These findings suggest that additional care should be taken when exhuming remains from older contexts.     

小鼠心肌细胞核DNA变化与死亡时间的关系

目的监测小鼠死后细胞核DNA降解的情况,探讨死后细胞核DNA降解的一般规律。方法应用单细胞凝胶电泳(SCGE)技术结合荧光显微镜和专业的计算机图像分析技术,测定111只小鼠在死后72h内不同时间点小鼠心肌组织细胞头半径、尾长度、头DNA含量比例、尾DNA含量比例、尾矩、Olive矩、头面积、尾面积八项参数的变化值。结果在个体死亡72h内,测定的八项参数指标均显示细胞核DNA降解速率和程度与死亡时间具有高度的相关性,并获得了更能体现DNA降解趋势的二项式回归方程和多元回归方程,均具有高度的统计学意义。结论应用72h内心肌组织DNA变化与死亡时间之间呈线性关系的各组回归方程,可为法医学推断死后经过时间提供一种新的、客观的、精确的方法和参考依据。     

Interleukin-6 and C-reactive protein serum levels in sepsis-related fatalities during the early postmortem period

Postmortem interleukin-6 (IL-6) and C-reactive protein (CRP) serum levels were investigated prospectively in sepsis-related fatalities and non-septic fatalities by using a linear regression model. At least three blood samples were collected between 0.3 and 139 h postmortem from sepsis-related fatalities (n=8) and non-septic fatalities (n=16). In addition, one antemortem blood sample was collected shortly before death from the septic patients. Antemortem and postmortem IL-6 and CRP levels were highly elevated in all individuals included in the sepsis group. An excessive postmortem increase of IL-6 serum levels associated with progressive time after death was observed in five out of the eight septic patients. Both, IL-6 and CRP serum concentrations seem to be suitable biochemical postmortem markers of sepsis. The determination of IL-6 serum levels above 1500 pg/ml in peripheral venous blood obtained in the early postmortem interval can be considered as a diagnostic hint towards an underlying septic condition. A more precise postmortem discrimination between sepsis and non-septic underlying causes of death is provided by the postmortem measurement of serum CRP in peripheral venous blood: on condition that at least two postmortem CRP values have been determined at different time points postmortem, the CRP level of a deceased at the time of death can be calculated by using linear regression analysis. When assessing postmortem IL-6 and CRP concentrations as biochemical postmortem markers of sepsis, various clinical conditions, such as a preceding trauma or burn injury going along with elevated IL-6 and/or CRP levels prior to death as a result of the systemic inflammatory response syndrome (SIRS) should be taken into consideration, thus adding relevant information for the practical interpretation of the results.     

IgE死后生化学检测的研究现状及应用展望

过敏性休克导致死亡的案例在法医学实践中常有发生,然而由于缺少特异的病理形态学指征,导致过敏性休克死亡的法医学鉴定面临一定的困难。IgE作为介导I型超敏反应的关键介质,其死后生化学检测对过敏性休克死亡案例的法医学鉴定具有辅助作用。但在实际应用中,血液等检材常受溶血等死后变化因素影响,尤其我国尸体保存以冷冻为主,溶血程度更加严重,对IgE等生化学指标的检测造成极大干扰。本文就目前IgE死后生化学检测的研究现状、生物学检材的选取及检测方法进行综述,并对现实应用中所面临的问题进行探讨。     

积温法则进行死亡时间推断

应用昆虫发育历期和积温法则推测死亡时间是法医学重要技术。本文对蝇蛆和蛹应用倒计积温的方法进行死亡时间推断,获得与实际死亡时间一致的结果。     

Distinguishing antemortem from postmortem injuries by LTB4 quantification

Leukotriene B₄ (LTB₄) in skin samples from seven forensic cases was detected by HPLC to distinguish their antemortem or postmortem origin. In total, there were thirteen antemortem and seven postmortem specimens. The results showed that LTB₄ was found in all antemortem wound specimens which were either fresh, or refrigerated or fixed in formalin for less than 10 days. In contrast, LTB₄ could not be detected in postmortem wound specimens. These results suggested that detecting the content of LTB₄ is a useful method for distinguishing antemortem from postmortem injuries.     

Preliminary study of the effect of acute antemortem haemorrhage on postmortem abdominal impedance in rats

Studies over many years have revealed a consistent, inverse relationship between extracellular impedance of the rat abdomen and postmortem interval (PMI). Recent studies have shown that this relationship can be improved by correcting measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C, thus rendering them independent of the tissues' temperature at the time of impedance measurement. The present study, undertaken as a part of an ongoing effort to identify variables which might influence postmortem rate of change in abdominal impedance, was aimed at studying the possible effect of acute antemortem haemorrhage on abdominal impedance. Sudden loss of approximately 30% of the total blood volume, immediately prior to the death was without statistically significant effect on the pattern of postmortem change in abdominal impedance. Thus, in the control and experimental rats, respectively, impedance decreased progressively from 58.7 +/- 1.8 and 68.0 +/- 11.3 omega, 1 h postmortem, to 31.5 +/- 2.2 and 33.7 +/- 4.7 omega at a postmortem interval of 120 h (n = 6 in each group). In both groups, the relationship between impedance and postmortem interval was either linear or curvilinear. These findings are believed to mitigate in favour of continued effort to examine the potential usefulness of abdominal impedance, as an index of postmortem interval, under conditions encountered in routine forensic investigation.     

STR typing of human DNA from fly larvae fed on decomposing bodies

In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.