CT analysis of skull contents in naturally mummified human corpses, a multicentric study.

INTRODUCTION: This study aimed to assess skull contents, brain appearance, and density on postmortem computed tomography in naturally mummified corpses. MATERIAL AND METHODS: For this purpose, a retrospective multicentric study, including mummified corpses from two French centers (Brest and Nantes) and from the New Mexico Decedent Image Database (USA), was performed by analyzing postmortem computed tomography (PMCT) focused on the head and neck of partially or fully mummified corpses discovered between 2011 and 2022. The PMCT analysis provided data on the CT appearance of brains, allowing them to be classified into four different categories (desiccation, liquefaction, dura mater only (DMO), and absence), and to measure densities (HU) of the brain remains. In addition, data on postmortem intervals (PMI) from Nantes and Brest centers were collected and analyzed to test the link between brain densities and PMIs. RESULTS: 54 cases of naturally mummified corpses were included. The brains were classified as liquefied (56%), desiccated (17 %), DMO (20 %), and absent (7 %) based on their CT appearance. Dehydrated brains were significantly (p < 0.004) denser (median 102 HU, interquartile range (IQR) 41) than either liquefied brains (median 39.5 HU, IQR 9) or brains with DMO (median -25 HU, IQR 57). However, the density of brain remains was not significantly affected by where the bodies were found (p = 0,41). Analysis of PMI and brain densities was performed on 22 cases. The results showed that brain remains were significantly (p = 0.039) denser when they were found after a PMI of more than six months. CONCLUSION: Brain desiccation was the aspect with the highest densities on PMCT, and for which we were able to highlight great preservation of anatomical structures observable in living organisms.

Postmortem sympathomimetic iris excitability.

BACKGROUND: A reliable estimation of time since death can be important for the law enforcement authorities. The compound method encompassing supravital reactions such as the chemical excitability of the iris can be used to further narrow intervals estimated by temperature-based methods. Postmortem iris excitability was mostly assessed by parasympatholytic or parasympathomimetic substances. Little is known regarding sympathomimetic agents. The present study aims to describe the postmortem iris excitability using the sympathomimetic drug phenylephrine. METHODS: Cadavers were included after body donors gave written informed consent during lifetime. Exclusion criteria were known eye disease, or a postmortem interval exceeding 26 hours. A pupillometer with a minimum measurement range of 0.5 mm was used to determine the horizontal pupil diameter before and 20 minutes after the application of phenylephrine. Increase in pupil diameter was labeled as positive reaction, unchanged pupil diameter was labeled as negative reaction, and decrease in pupil diameter was labeled as paradox reaction. RESULTS: 30 eyes from 16 cadavers (median age = 80.0; 9 males, 7 females) were examined. Initial pupil size was in median 3.5 mm (interquartile range [IQR]: 3.0-4.5 mm) and progressed to 4.0 mm (IQR: 3.5-5.0 mm) 20 minutes after drug instillation. The achieved pupil diameter difference comprised in median 0.5 mm (IQR: 0.0-1.0 mm). A positive reaction was observed in 21 cases. Negative reactions were observed in 5 cases and paradox reactions in 4 cases. Overall, there was a statistically significant difference in diameter between the initial and the reactive pupil (P = 0.0002). CONCLUSION: Although relatively rarely used, sympathomimetic drugs seem to be eligible for chemical postmortem iris excitability. Currently, assessment of postmortem iris excitability usually only involves parasympatholytic and parasympathomimetic agents. The findings of the present study give a hint that the application of a third agent with a sympathomimetic mechanism of action could provide additional information. Further studies assessing such a triple approach in the compound method in comparison with the current gold standard for estimation of time since death are mandatory to ensure reliable results.

Improving accuracy of age estimates for insect evidence-calibration of physiological age at emergence (k) using insect size but without "k versus size" model.

Postmortem interval may be estimated based on the age of insect evidence collected on a death scene. Reference data that are used in such estimation frequently comprise thermal summation constant (i.e. k), which is equal to the insect age upon completion of immature development expressed in accumulated degree-days or degree-hours (ADD or ADH). Essentially, k is a central point of an insect group and it may poorly represent insect evidence that is near the limits of variation for the group. Accordingly, it was postulated to calibrate k for particular insect evidence and insect size and sex were found to be useful for this purpose in some of the species. However, the calibration is only possible by using the model that correlates k with insect size. Since very few such models were published, this lack of data limits the calibration of k in forensic casework. In this article, we develop a formula that is useful for the calibration of k without the use of "k versus size" model (and related datasets). The formula uses k from the general thermal summation model for a species (with its standard error), size range for the species (retrieved from entomology literature), and size measurements for particular insect evidence. The calibration of k with the formula was validated using the Creophilus maxillosus (Coleoptera: Staphylinidae) and Necrodes littoralis (Coleoptera: Silphidae) datasets. It was particularly useful while analyzing unusually small and large insects, in case of which the formula reduced the inaccuracy of k from the general model on average by ~25 ADD in C. maxillosus and ~40 ADD in N. littoralis. We discuss the limitations and prospects of the calibration protocol that employs the formula.

The micro CT evaluation of crown and root pulp volume versus dentin thickness in teeth in postmortem interval (PMI).

Determining the postmortem interval (PMI) is one of the main study subjects of forensic sciences. The main purpose of this prospective in vitro study that was the Micro-CT evaluation of teeth crown and root pulp volume versus dentin thickness in terms of PMI determination. The study involved 60 female Wistar rats, with weights ranging from 270 to 320 g. These rats were grouped into six different post-mortem period categories. Following the animals' sacrifice, they were subjected to a natural putrefaction period, with a control group, in the grounds of a sheltered garden. Hemi-mandible samples were then extracted and placed in glass tubes for Micro-CT evaluations, following the progression of putrefaction processes. The pulp volume and dentin thickness were assessed using Micro-CT, and the gathered data underwent statistical analysis. Micro-CT was employed to analyze sixty right mandibular second molar teeth in the hemi-mandible. The crown pulp volume exhibited a reduction in group 6, with a value of 0.239 mm3 after a three-month period of natural putrefaction (p < 0.001). There is statistically differences among groups in case of pairwise comparison (p < 0.05). However, the root pulp volume and dentin thickness variables did not display any statistically significant changes. Despite certain limitations associated with this study, the Micro-CT findings concerning teeth pulp volume can serve as an objective parameter, especially for late postmortem investigations and the estimation of time of death.

Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Artificial intelligence-driven microbiome data analysis for estimation of postmortem interval and crime location.

Microbial communities, demonstrating dynamic changes in cadavers and the surroundings, provide invaluable insights for forensic investigations. Conventional methodologies for microbiome sequencing data analysis face obstacles due to subjectivity and inefficiency. Artificial Intelligence (AI) presents an efficient and accurate tool, with the ability to autonomously process and analyze high-throughput data, and assimilate multi-omics data, encompassing metagenomics, transcriptomics, and proteomics. This facilitates accurate and efficient estimation of the postmortem interval (PMI), detection of crime location, and elucidation of microbial functionalities. This review presents an overview of microorganisms from cadavers and crime scenes, emphasizes the importance of microbiome, and summarizes the application of AI in high-throughput microbiome data processing in forensic microbiology.

The Role of Protein Degradation in Estimation Postmortem Interval and Confirmation of Cause of Death in Forensic Pathology: A Literature Review.

It is well known that proteins are important bio-macromolecules in human organisms, and numerous proteins are widely used in the clinical practice, whereas their application in forensic science is currently limited. This limitation is mainly attributed to the postmortem degradation of targeted proteins, which can significantly impact final conclusions. In the last decade, numerous methods have been established to detect the protein from a forensic perspective, and some of the postmortem proteins have been applied in forensic practice. To better understand the emerging issues and challenges in postmortem proteins, we have reviewed the current application of protein technologies at postmortem in forensic practice. Meanwhile, we discuss the application of proteins in identifying the cause of death, and postmortem interval (PMI). Finally, we highlight the interpretability and limitations of postmortem protein challenges. We believe that utilizing the multi-omics method can enhance the comprehensiveness of applying proteins in forensic practice.

Evaluation of porcine decomposition and total body score (TBS) in a central European temperate forest.

The total body score (TBS) is a visual scoring method to scale the succession of decomposition stages. It compares decomposition between cadavers, to connect it with external taphonomic factors and estimate the post-mortem interval. To study decomposition in various climatic environments, pigs are often used as human proxies. Currently, there is one TBS system by Keough et al. (J Forensic Sci. 2017;62:986) for surface-deposited domestic pigs, coming from South Africa. Our study aims to evaluate this method and analyze porcine decomposition in Central Europe to inform forensic research and casework. We conducted an experiment studying six 50 kg pig carcasses in a temperate Swiss forest. Three observers documented decomposition patterns and rated the decomposition stages from photographs based on the porcine TBS model by Keough et al. (J Forensic Sci. 2017;62:986). We documented discrepancies between the carcass decomposition of our specimens and those in the South African study, especially related to the high insect activity in our experiment. Furthermore, we noted factors complicating TBS scoring, including rainfall and scavengers. The agreement between TBS observers from photographs was in the highest agreement category apart from one "substantial agreement" category. Our study is the first in Europe to systematically test the Keough et al. (J Forensic Sci. 2017;62:986) method. The results evidence that regional adaptations are required to be applicable for other environments. We present a modified approach based on experimental observations in a Swiss temperate forest. The identification of regional decomposition patterns and drivers will inform future taphonomy research as well as forensic casework in comparable contexts in Central Europe.

Short-term postmortem interval estimation by detection of apoptosis-related protein in skin.

Time-of-death extrapolation has always been one of the most important issues in forensic practice. For a complicated case in which a corpse is destroyed with little evidence, judging the time of death of the deceased is a major challenge, which also enables criminals to escape legal sanctions. To find a method to roughly judge the time of death of a corpse with only a small amount of skin tissue, in this study, we established an early death model by using mice; furthermore, the postmortem interval was estimated by determining the protein and mRNA levels of Bax and Bcl-2 in the skin. In this process, 0 h after death was used as the control group, and the expression levels of Bax and Caspase-3 reached the maximum value at 8-12 h, while Bcl-2, as an inhibitor of apoptosis protein, peaked after 24 h. The mRNA expression levels of related proteins in postmortem skin tissues were also different. The results of these data indicate that the protein and mRNA levels of Bax and Bcl-2 in the skin have potential application in early time-of-death estimation.

Development of Megaselia scalaris at constant temperatures and its significance in estimating the time of death.

Megaselia scalaris (Schmitz, 1938) (Diptera: Phoridae) is a common insect in forensic science that is frequently found in indoor cases, particularly on corpses in closed environments. Although this species is useful for estimating the minimum postmortem interval (PMImin) in the absence of Calliphoridae, there is a lack of data on its development in China. Herein, we studied the development of M. scalaris exposed to seven constant temperatures ranging from 16 to 34 °C. The mean (± SD) developmental durations of M. scalaris from egg to adult stage at 16, 19, 22, 25, 28, 31, and 34 °C were 1486.9 ± 75.3, 823.7 ± 42.8, 448.2 ± 59.8, 417.7 ± 19.7, 297.2 ± 27.3, 272.9 ± 10.4, and 253.0 ± 5.0 h, respectively. The mean (± SE) lower developmental threshold temperature (TL) and the thermal summation constant (K) were determined by a linear model as 12.69 ± 0.3 °C and 4965.8 ± 227.9-degree hours, respectively. A nonlinear model estimated the lower developmental threshold temperature, intrinsic optimum temperature, and upper lethal developmental threshold temperature as 14.58, 21.00, and 34.15 °C, respectively. We established three development models to estimate the age of the immature insect, namely the isomegalen diagram, isomorphen diagram, and thermal summation model. In addition, a regression analysis of the relationship between body length and total development time from hatching to pupariation was performed. Our findings provide a basis for applications of M. scalaris in PMImin estimations.

The relationship between postmortem interval and protein changes in mice.

Postmortem interval (PMI) estimation is important for forensic pathological autopsy. It has been reported that there is a correlation between certain protein changes in cadavers and PMI. However, no specific protein(s) has been used to determine the PMI so far. In this study, the total protein contents of mouse liver and spleen at different time of death were measured. The data showed that they were negatively correlated with the PMI. The degradation of ß-actin was found to be positively correlated with the PMI in the liver. Additionally, proteomic technique was used to study the changes of protein expression related to PMI in the liver of mice. By using Two-dimensional electrophoresis, the expressions of four proteins were found to be significantly decreased and those of other three proteins were unchanged with the increase of PMI. Among the seven proteins, six were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The three altered proteins were SBP2, ENOA, ALDH2 and three unchanged ones were 3HAO, TPIS, CATA, respectively. In the future, those unchanged proteins could be used as internal references to more accurately and reliably infer the time of death by assessing the level of changed proteins.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

[Mathematical simulation of the corpse's temperature change during exposure of a direct solar radiation to diagnose a postmortem interval]. / Matematicheskoe modelirovanie protsessa izmeneniya temperatury trupa pri vozdeistvii na nego pryamoi solnechnoi radiatsii v tselyakh diagnostiki davnosti smerti.

OBJECTIVE: To choose an optimal mathematical model and to practice the method of its use during finding the corpse under exposure of a direct solar radiation. MATERIAL AND METHODS: The studies were carried out on 9 corpses with body weight from 50 to 83 kg. The influence of black (6 corpses) and white (3 corpses) colored cotton fabrics was investigated to standardize a photoreceptive surface of clothes. In addition, the objects (bags made of substantial polyethylene, densely filled with moderately humid black soil) replacing corpses with mass of 5.5, 11.0, 16.5 and 22.0 kg, wrapped in cotton fabric of specified colors, that imitates clothes, were used. The objects' temperature was measured within the accuracy of 0.001 °C, air temperature - within accuracy of 0.1 °C. A simplified one-dimensional variant of the finite mathematical differences method, representing the process of corpse's cooling as a discrete transfer of heat from one layer (node) to another, was used in developing the mathematical modeling algorithm. The model consisted of ten nodes was used in calculations. RESULTS: A mathematical model in the form of equations' system was developed, in which the variable reflecting the intensity of corpse's surface heating by sun rays was introduced. The example of calculation made with data of real measurements of corpse's temperature, that was in open place for the sun rays, is shown.

Development of Phormia regina at seven constant temperatures for minimum postmortem interval estimation.

Phormia regina (Meigen, 1826) (Diptera: Calliphoridae) can colonize carcasses quickly, and its immature stages are reliable entomological evidence for the estimation of the minimum postmortem interval (PMImin). There are discrepancies in the developmental data from previous studies on P. regina, and the related PMImin indicators need to be refined. We investigated the accuracy of forensic entomological evidence using development durations, growth accumulated degree hours, and larval body length variations of P. regina at seven constant temperatures ranging from 16 to 34 °C. We also established development models such as the isomorphen diagram, thermal summation model, isomegalen diagram, and body length simulation equation to assist with PMImin estimation. The developmental duration of P. regina from egg to adult at 16, 19, 22, 25, 28, 31, and 34 °C was 840.8 ± 42.8 h, 580.1 ± 10.1 h, 390.4 ± 8.7 h, 316.8 ± 9.4 h, 291.4 ± 21.2 h, 238.4 ± 2.8 h, and 222.5 ± 5.2 h, respectively. The lower threshold temperature TL was 9.97 ± 0.50 °C, while the thermal constant K was 5052.7 ± 229 degree days. The lower developmental thresholds, intrinsic optimum temperature, and upper lethal developmental threshold obtained by the Optim SSI models were 13.15, 21.20, and 36.86 °C, respectively. This study aims to provide developmental models for P. regina aimed at common case-site temperatures in the northern provinces of China, which can be used for accurate PMImin estimation.

Luminol and the postmortem interval estimation - influence of taphonomic factors.

The estimation of the postmortem interval (PMI) is one of the key challenges for forensic anthropologists. Although there are several methods referenced for this purpose, none is sufficiently effective. One of the main reasons justifying the complexity of this task is the influence of several taphonomic factors.The study of the Luminol technique has stood out as a promising method for estimating PMI, complementing the existing methods, since it is an economic, easy and reproducible method that operates as a presumptive test. However, it is not known which taphonomic factors can influence the results obtained by this technique.The aim of this study is to test the influence of taphonomic factors, such as temperature, humidity, soil type and pH, on the estimation of the PMI by the Luminol technique.In order to test the influence of the referred factors, a sample consisting of 30 clavicles, with known and similar PMI, collected from autopsies, was distributed as evenly as possible by six vases and buried with different decomposition conditions for a period of 12 months. After the exhumation and sample preparation, the Luminol technique was applied.It was possible to clearly observe differences in the results. Thus, according to our research, it is possible to conclude that the results obtained by the application of Luminol are influenced by taphonomic factors. Therefore, the context in which a body is found should always be considered for applying this technique.

Changes in Microbial Communities Using Pigs as a Model for Postmortem Interval Estimation.

Microbial communities can undergo significant successional changes during decay and decomposition, potentially providing valuable insights for determining the postmortem interval (PMI). The microbiota produce various gases that cause cadaver bloating, and rupture releases nutrient-rich bodily fluids into the environment, altering the soil microbiota around the carcasses. In this study, we aimed to investigate the underlying principles governing the succession of microbial communities during the decomposition of pig carcasses and the soil beneath the carcasses. At early decay, the phylum Firmicutes and Bacteroidota were the most abundant in both the winter and summer pig rectum. However, Proteobacteria became the most abundant in the winter pig rectum in late decay. Using genus as a biomarker to estimate the PMI could get the MAE from 1.375 days to 2.478 days based on the RF model. The abundance of bacterial communities showed a decreasing trend with prolonged decomposition time. There were statistically significant differences in microbial diversity in the two periods (pre-rupture and post-rupture) of the four groups (WPG 0-8Dvs. WPG 16-40D, p < 0.0001; WPS 0-16Dvs. WPS 24-40D, p = 0.003; SPG 0D vs. SPG 8-40D, p = 0.0005; and SPS 0D vs. SPS 8-40D, p = 0.0208). Most of the biomarkers in the pre-rupture period belong to obligate anaerobes. In contrast, the biomarkers in the post-rupture period belong to aerobic bacteria. Furthermore, the genus Vagococcus shows a similar increase trend, whether in winter or summer. Together, these results suggest that microbial succession was predictable and can be developed into a forensic tool for estimating the PMI.

Estimation of postmortem interval using histological and oxidative biomarkers in human bone marrow.

In forensic medicine, estimating the postmortem interval (PMI) is of great importance for the timeline and the reconstruction of the events surrounding death. Bone marrow (BM) is one of the largest organs in the body, with good resistance to autolysis and contamination. Therefore, the present study aims to correlate different postmortem intervals and bone marrow antioxidant enzyme levels using an Enzyme-linked immunosorbent assay (ELISA). In addition, detection of the changes in the histological structure of human bone marrow in relation to the time passed since death. BM samples from 20 forensic autopsy cadavers were obtained from cases referred to the Department of Forensic Medicine in the Ministry of Justice, Dakahlia Governorate, processed for histopathological examination as well as estimation of reduced glutathione (GSH), glutathione peroxidase (GPX), and glutathione reductase (GRX) using ELISA. Results of ELISA analysis showed a significant decrease in the level of antioxidant enzymes with increasing PMI; regarding histopathological examination, from 6 to > 18 h PMI, the changes in morphology after death were gradual, progressive, and regular, indicating great value in PMI determination. Also, 18 h of PMI showed loss of cellular details, absence of fat cells, and necrosis of BM with the nucleus dispersed as eosinophilic debris. Estimation of antioxidant enzymes level in human bone marrow using ELISA and detection of the changes in the histological structure of human bone marrow in relation to time passed since the death, either separately or in combination, can be used to estimate PMI accurately.

A computational approach to estimate postmortem interval using postmortem computed tomography of multiple tissues based on animal experiments.

The estimation of postmortem interval (PMI) is a complex and challenging problem in forensic medicine. In recent years, many studies have begun to use machine learning methods to estimate PMI. However, research combining postmortem computed tomography (PMCT) with machine learning models for PMI estimation is still in early stages. This study aims to establish a multi-tissue machine learning model for PMI estimation using PMCT data from various tissues. We collected PMCT data of seven tissues, including brain, eyeballs, myocardium, liver, kidneys, erector spinae, and quadriceps femoris from 10 rabbits after death. CT images were taken every 12 h until 192 h after death, and HU values were extracted from the CT images of each tissue as a dataset. Support vector machine, random forest, and K-nearest neighbors were performed to establish PMI estimation models, and after adjusting the parameters of each model, they were used as first-level classification to build a stacking model to further improve the PMI estimation accuracy. The accuracy and generalized area under the receiver operating characteristic curve of the multi-tissue stacking model were able to reach 93% and 0.96, respectively. Results indicated that PMCT detection could be used to obtain postmortem change of different tissue densities, and the stacking model demonstrated strong predictive and generalization abilities. This approach provides new research methods and ideas for the study of PMI estimation.

Research Progress on Microbial Community Succession in the Postmortem Interval Estimation.

The postmortem interval (PMI) estimation is a key and difficult point in the practice of forensic medicine, and forensic scientists at home and abroad have been searching for objective, quantifiable and accurate methods of PMI estimation. With the development and combination of high-throughput sequencing technology and artificial intelligence technology, the establishment of PMI model based on the succession of the microbial community on corpses has become a research focus in the field of forensic medicine. This paper reviews the technical methods, research applications and influencing factors of microbial community in PMI estimation explored by using high-throughput sequencing technology, to provide a reference for the related research on the use of microbial community to estimate PMI.

Usability of elemental analysis for antemortem and postmortem bone fracture differentiation.

The differentiation of antemortem and postmortem fractures is a challenging and important issue in forensic medicine. To investigate the usability of chemical element levels in blunt fracture ends for the differentiation of antemortem and postmortem fractures, antemortem and postmortem fractures were created in rats and left to decay in the natural environment. Forty-eight rats were used in this study. The rats divided into six groups, each group containing eight rats. We induced a left mandibular fracture in all rats under anesthesia and then waited for 2 d to become organized to fracture the hematoma. All rats were sacrificed under anesthesia, and a postmortem blunt fracture was created in the right mandible. Samples were taken from the fracture ending at 0, 7, 14, 30, 60, and 90 d after the blunt postmortem fracture. The Mg, S, K, Ca, and Fe contents were measured using inductively coupled plasma/optical emission spectrometry (ICP-OES). In our study, it was observed that antemortem Mg, K, S and Ca levels were generally higher than the post-mortem levels, although not statistically significant in all groups.