The Fascinating World of Forensic Sciences: Multidisciplinary Approaches in Human Remains and the Role of Forensic Pathology in Calabrian Experience.

Forensic sciences play a vital role in the criminal justice system by providing insights into the identity of victims and suspects, causes of death, and other crucial pieces of evidence. In this research paper, we will explore the utility of forensic sciences, its techniques and applications, and the critical role of the forensic pathologist in analyzing human remains. For this purpose, we have analyzed a series of human remains and cadavers found in a state of decomposition, illustrating the medico-legal investigations carried out. Specifically, 50 cases from Calabrian experience are reported from 2003 to 2023 in different contexts of both judicial and archaeological interest and discovered by chance. In any case, anthropological, odontological, genetic, entomological, and forensic radiological investigations were carried out with the supervision of the forensic pathologist in all cases. The varied composition of the sample made it possible to understand the methods and the various specialists involved in such diversified cases. Furthermore, a review of the scientific literature on the topic of human remains was carried out. In particular, by delving into these topics, we aim to provide a comprehensive understanding of forensic anthropology and forensic sciences and their significance in the criminal justice system.

Microscopic fungi on the corpse - Promising tool requiring further research.

Forensic microbiology is a relatively new area of forensic sciences. It considers the potential of microorganisms to be used in criminal investigations. As most studies involve the role of bacteria in fields like post-mortem interval estimation, personal identification or geolocation, the data on the role of fungi is comparatively scarce. Forensic mycology involves the application of fungi and their structures in forensic cases. The aim of this review is the evaluation of the current state of knowledge on fungi associated with human cadavers and their possible role in estimating the time since death. In accordance with the available reports, we focused on the relation between microscopic fungi isolated from human corpses and the cadaver condition e.g., the stage of decomposition. We also emphasised the contrast between the reported methodologies and attempted to standardise research methods in forensic mycology from sample collection to its storage, mycological analysis and identification of the obtained fungal cultures. Moreover, the potential usage of microscopic fungi in criminal cases was discussed based on various case reports.

Development and intrapuparial characterization of Peckia (Euboettcheria) collusor (Curran and Walley, 1934) (Diptera: Sarcophagidae) for application in forensic entomology.

The family Sarcophagidae is very diverse in Brazil. Due to their living habits, they are the subject of many medical, veterinary, sanitary, and entomological studies. However, Sarcophagidae species are still poorly studied in forensic entomology, although they are frequently reported in carcasses and even human corpses. Thus, this study aims to identify and compare the developmental stages and intrapuparial morphological characteristics of Peckia (Euboettcheria) collusor to serve as an auxiliary tool in forensic entomology. The pupae collected after zero hour at 27 °C and 32 °C were sacrificed every three hours until the first 24 h and then every six hours until the emergence of the first adults, using 30 pupae each time, totaling 1560 for 27 °C and 1290 for 32 °C. The intrapuparial development time of this fly species under laboratory-controlled conditions was 288 h at 27 °C and 228 h at 32 °C. The 2820 pupae were analyzed according to temperature and classified into eight possible stages. This contributed to the selection of 16 key morphological characteristics to identify the age of the pupae. The identified intrapupal morphological characteristics have great potential to help researchers, experts, technical assistants, and forensic entomologists estimate the minimum post-mortem interval (minPMI) of cadavers.

The influence of cooling on biomechanical time since death estimations using ovine brain tissue.

The significance of biomechanical analyses for forensic time since death estimations has recently been demonstrated. Previous biomechanical analyses successfully discriminated post-mortem brain tissue from tissue with a post-mortem interval of at least one day when held at 20 °C. However, the practical utility of such analyses beyond day one at 20 °C was limited. This study investigates the storage, loss, and complex shear modulus of various brain regions in sheep stored at 4 °C in 24-hour intervals over four days post-mortem using rheometry tests. The aim is to identify the critical biomechanical tissue property values to predict post-mortem time and assess the temperature sensitivity of the rheometry method by comparing results to recent findings at 20 °C. Thirty sheep brains were examined, including the frontal lobe, parietal lobe, anterior and posterior deep brain, superior colliculi, pons, medulla, and cerebellum. Rheometry tests were conducted, and receiver operator characteristic analyses were employed to establish cut-off values. At 4 °C storage, all investigated biomechanical properties of the examined brain regions remained stable for at least one day post-mortem. Using cerebellar samples stored at 4 °C, a post-mortem interval of at least two days could be determined with excellent diagnostic ability. Complex shear modulus values below 1435 Pa or storage modulus values below 1313 Pa allowed prediction of two or more days post-mortem. Comparisons between 4 °C and 20 °C revealed brain region-specific results. For instance, the complex shear moduli of the anterior deep brain at 4 °C were significantly higher on all individual testing days when compared to 20 °C. In contrast, the combined medulla and pons samples were similar on each day. Rheometry testing of brain tissue consistently stored at 4 °C since death proved valuable for forensic time since death estimations starting from two days after death.

The influence of rehydration on decomposition in the Highveld region of South Africa-Using a pig model.

Researchers have observed that rainfall may re-initiate decomposition in desiccated tissue; however, no conclusive research-based evidence exists on the specific effects of rehydration on decomposition. Therefore, this study aimed to assess the effects of artificial rehydration on the progression of decomposition following the advanced stage of decomposition. Twelve adult pig cadavers (8 experimental; 4 controls) were placed in the central Highveld of South Africa during cooler (April-July 2021) and warmer (August-November 2021) months. Decomposition was scored approximately biweekly to obtain the total body score, and accumulated degree days (ADD) were calculated for each pig. All pig cadavers were covered by chicken wire cages with transparent tarps to control for natural rehydration and scavenging. Once the experimental pig cadavers reached a three-visit stasis in the advanced phase of decomposition, they were artificially rehydrated, and changes in the progression of decomposition between the control and experimental groups were plotted (ADD against TBS) for observation. The rehydrated experimental pig cadavers showed re-initiation of decay and insect re-colonization, while the control cadavers mainly remained in a state of stasis with insect activity ceased altogether. Greater cadaver decomposition islands and a color change post-rehydration were also noted in some experimental cadavers. This supports the need for future research on the impact of rehydration, including associated soil moisture on decomposition rates, progression, and invertebrate colonization, which will enhance our understanding of the effects these environmental factors have on the accuracy of post-mortem interval estimation.

Carrion insects living within the bones of large mammals: insect conservation and forensic entomology implications.

Succession patterns of carrion insects on large mammal's carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone's inner cavity, was monitored with a camera to assess the insect's putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

Detrimental effects of scene manipulations on temperature-based time since death estimation.

In forensic casework, time since death (TSD) estimations may play a crucial role to establish chains of events as well as for alibi assessment in homicide cases. Classical TSD estimation relies on reasonably stable ambient temperatures and a correct documentation of ambient and rectal temperatures. This constancy is in some cases disturbed by post-discovery alterations of the crime scene, e.g. opening a window. In order to develop a better understanding of this alteration-based detrimental impact on TSD estimation as well as to identify feasible recommendations for casework, the present pilot study examined ambient temperature effects of different window opening scenarios regarding various time intervals (5 to 360 min) in a furnished 10 m2 apartment during winter. In this context, in addition to the ambient temperature and thus the cooling rate of the room, re-approximation to initial room temperature, potential influences on a nomogram-based time since death estimation using a fictitious case, and the impact of the measurement height above the ground were investigated. Our data indicate a significant reduction of the mean temperature decrease rate after 15 min regardless of the remaining opening time and a correlation with the size of the respective opening surfaces. Re-approximation to initial room temperatures was observed with up to three times longer than the initial opening time. There was no evidence of a substantial advantage of temperature measurements above the level of the corpse (> 0.1 m). The limitations of the study and its applicability for forensic casework are critically reviewed.

Counting the days: Exploring the post-mortem interval factors in sexual homicides.

This study explores the offender, victim, and environmental characteristics that significantly influence the number of days a sexual homicide victim remains undiscovered. Utilizing a sample of 269 cases from the Homicide Investigation Tracking System database an in-depth analysis was conducted to unveil the factors contributing to the delay in the discovery of victims' bodies. The methodological approach involves applying a negative binomial regression analysis, which allows for the examination of count data, specifically addressing the over-dispersion and excess zeros in the dependent variable - the number of days until the victim is found. The findings reveal that certain offender characteristics, victim traits, and spatio-temporal factors play a pivotal role in the time lag experienced in locating the bodies of homicide victims. These findings have crucial implications for investigative efforts in homicide cases, offering valuable insights that can inform and enhance the efficacy and efficiency of future investigative procedures and strategies.

From flesh to bones: Multi-omics approaches in forensic science.

Recent advancements in omics techniques have revolutionised the study of biological systems, enabling the generation of high-throughput biomolecular data. These innovations have found diverse applications, ranging from personalised medicine to forensic sciences. While the investigation of multiple aspects of cells, tissues or entire organisms through the integration of various omics approaches (such as genomics, epigenomics, metagenomics, transcriptomics, proteomics and metabolomics) has already been established in fields like biomedicine and cancer biology, its full potential in forensic sciences remains only partially explored. In this review, we have presented a comprehensive overview of state-of-the-art analytical platforms employed in omics research, with specific emphasis on their application in the forensic field for the identification of the cadaver and the cause of death. Moreover, we have conducted a critical analysis of the computational integration of omics approaches, and highlighted the latest advancements in employing multi-omics techniques for forensic investigations.

An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Catch me if you can-emission patterns of human bodies in relation to postmortem changes.

The present study examines for the first time the emission patterns and olfactory signatures of 9 complete human corpses of different stages of decomposition. Air sampling was performed inside the body bags with solid sorbents and analysed by coupled gas chromatography-mass spectrometry after thermal desorption (TD-GC-MS). Furthermore, odour-related substances were detected by gas chromatography-olfactometry (GC-O). Sulfurous compounds (mainly dimethyl di- and trisulfide) were identified as most important to the odour perception. Around 350 individual organic substances were detected by TD-GC-MS, notably sulfurous and nitrogenous substances as well as branched alkanes, aldehydes, ketones, alcohols, carboxylic acids, carboxylic acid esters and ethers. A range of terpenes was detected for the first time in a characteristic emission pattern over all decomposition stages. Concentrations of the substances varied greatly, and no correlation between the emission patterns, the stage of decomposition and the cause of death could be found. While previous studies often analysed pig cadavers or only parts of human tissue, the present study shows the importance of analysing complete human corpses over a range of decomposition stages. Moreover, it is shown that using body bags as a kind of "emission test chamber" is a very promising approach, also because it is a realistic application considering the usual transport and store of a body before autopsy.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Protophormia terraenovae (Diptera: Calliphoridae).

Protophormia terraenovae is a colonizer of decomposing bodies and is known to cause pre-mortem myiasis as the female flies lay eggs in uncleaned wounds. In this study the effects of different concentrations of antibiotics levofloxacin and ceftriaxone on maggot development, weight, length, and mortality were examined. The maggot length and weight were significantly increased by therapeutical doses of levofloxacin and ceftriaxone. The maggot development time was significantly decreased in every levofloxacin treatment compared to the control. The time to start pupation was significantly increased in the control compared to the antibiotic treatments. Levofloxacin significantly increased the survivability of the maggots. Every levofloxacin treatment significantly improved the rearing conditions for the maggots. Reaching the third instar was delayed by 24 h in the control compared to the Levo 3.57 treatment. The Pupation in the control was delayed by an average of 48 h compared to the Levo 3.57 treatment. The significantly reduced development time of the maggots in the antibiotic treatments might lead to an overestimation of the post-mortem interval and therefore an incorrect time of death determination. The improved rearing conditions may be an indication of the potential of a combined application of antibiotics and maggot therapy.

Volabolomic Fingerprinting for Post-Mortem Interval Estimation: A Novel Physiological Approach.

Death is a multifaceted process wherein each individual cell and tissue has a metabolic homeostasis and a time of functional cessation defined by the dying process as well as by intrinsic and extrinsic factors. Decomposition is physiologically associated with the release of different types of volatile organic compounds (VOCs), and these form volaboloma mortis. The main purpose of this study was to record the volabolomic fingerprint produced by volatile molecules during the physiological decomposition process of human tissue and muscle cells. The volatile chemical signature has important implications for an open issue in forensics and pathology, namely the estimation of the postmortem interval (PMI), which decreases in accuracy with the passage of time. Volatile metabolites emitted from human tissues and muscle cells at 0, 24, 48, and 72 h were recorded in real time with an electronic nose sensor device. The key findings were the continuous sampling of VOCs emitted from tissues and cells. These showed a common behavior as time progressed; particularly, after 48 h the distributions became dispersed, and after 72 h they became more variable. Volabolomic fingerprinting associated with time progression relevant to the study of PMIs was reconstructed. Additionally, there may be broader applications, such as in dog training procedures for detecting human remains, and perhaps even for studying scavenger and insect attractants.

Estimation of the post-mortem interval; added value of mechanical excitability of human skeletal muscle.

Estimation of the post-mortem interval (PMI) is mainly based on the state of body cooling, post-mortem lividity (livor mortis) and post-mortem muscle stiffness (rigor mortis). However, the time span of development of these post-mortem phenomena are influenced by a variety of factors concerning the body of the deceased and the environment in which the body is found. Subsequently, this leads to a substantial spread in upper and lower limits of PMI based on determination of the state of these phenomena. Moreover, interpretation of post-mortem phenomena like lividity, rigor and interpretation of the correction factor for Henssge's nomogram is subjective. For this reason, PMI estimations are often broad, possibly too broad to be helpful for answering questions which are relevant for the criminal investigation. Therefore, combining the outcome of different methods for estimating the PMI, the so-called compound method, is recommended. Supravital muscle reaction by mechanical stimulation of skeletal muscle is a less known aspect of the compound method. Here we present a series of cases series in which supravital muscle reaction contributed to a more precise estimation of the PMI.

Forensic taphonomic experimental design matters: a study assessing clothing and carrion biomass load on scavenging in Cape Town, South Africa.

The identification of unknown human remains is a significant and ongoing challenge in South Africa, worsened by the country's high murder rate. The rate of decomposition in South Africa is significantly influenced by vertebrate scavenging, which, if not considered, can impede the accurate estimation of the post-mortem interval. Scavenging patterns vary greatly depending on the environment and ecological region, and there is limited data for the Western Cape province. To address this gap, two clothed and uncaged pig carcasses weighing 60 kg each were placed in the field in July 2021 and January 2022, respectively. Motion-activated infrared-capable trail cameras were used to observe decomposition, scavenger species, and their activities. Additionally, a comparative sample of 16 unclothed carcasses deployed between 2014 and 2016 in the same habitat were analyzed to assess the impact of clothing and biomass load. The study found three main results: (1) Regardless of habitat or biomass load, it took significantly less time to reach decomposition milestones (25%, 50%, and 75%) during the summer season; (2) the presence of mongoose scavengers had a greater impact on the time required to reach milestones during winter compared to summer; and (3) single carcass deployments reached the milestones faster than multi-carcass deployments in both seasons. This research highlights the potential inaccuracy of current methods for estimating the post-mortem interval when scavenging activity is not considered or documented in the underlying experimental data, particularly for environments or ecological biomes where scavengers actively impact decomposition rates.

Handheld hyperspectral imaging as a tool for the post-mortem interval estimation of human skeletal remains.

In forensic medicine, estimating human skeletal remains' post-mortem interval (PMI) can be challenging. Following death, bones undergo a series of chemical and physical transformations due to their interactions with the surrounding environment. Post-mortem changes have been assessed using various methods, but estimating the PMI of skeletal remains could still be improved. We propose a new methodology with handheld hyperspectral imaging (HSI) system based on the first results from 104 human skeletal remains with PMIs ranging between 1 day and 2000 years. To differentiate between forensic and archaeological bone material, the Convolutional Neural Network analyzed 65.000 distinct diagnostic spectra: the classification accuracy was 0.58, 0.62, 0.73, 0.81, and 0.98 for PMIs of 0 week-2 weeks, 2 weeks-6 months, 6 months-1 year, 1 year-10 years, and >100 years, respectively. In conclusion, HSI can be used in forensic medicine to distinguish bone materials >100 years old from those <10 years old with an accuracy of 98%. The model has adequate predictive performance, and handheld HSI could serve as a novel approach to objectively and accurately determine the PMI of human skeletal remains.

Morphometric analysis for determination of larval instars in Dermestes frischii Kugelann and Dermestes undulatus Brahm (Coleoptera: Dermestidae).

Dermestes frischii Kugelann, 1792 and Dermestes undulatus Brahm, 1790 are the most abundant species worldwide at outdoor or indoor crime scenes during the dry and skeletal stages of decomposition. The attribution of larval age in these beetles is problematic due to the variable number of instars, which is influenced by environmental factors. In this study, a morphometric approach was used to look for potential morphological features as evidence of larval stages. Breeding and monitoring were performed for both species in an incubator with a preset temperature of 28°C ± 0.5 without a photoperiod. Morphometric measurements were made on 10 larvae per instar for each species using length, width, and thickness parameters. Linear discriminant analysis was then used to generate decision boundaries that clearly separated larval stages. The cross-validation procedure demonstrated that the morphometric approach successfully discriminated adjacent larval stages in both species with high values of sensitivity and specificity. This less-invasive approach could improve the ability to estimate minPMI in forensic studies of Dermestidae beetles. Future studies may extend this approach to other species and establish good practices for collecting and storing specimens for morphometric analysis.

Immunohistochemical expression of HMGB1 and related proteins in the skin as a possible tool for determining post-mortem interval: a preclinical study.

Determining the post-mortem interval (PMI) is one of forensic pathology's primary objectives and one of its most challenging tasks. Numerous studies have demonstrated the accuracy of histomorphology and immunohistochemical investigations in determining the time of death. Nevertheless, the skin, a robust and easy-to-remove tissue, has only been partially analyzed so far. By studying 20 adult male mice, we tried to determine whether post-mortem immunohistochemical detection in the skin of HMGB1 proteins and associated components (Beclin1 and RAGE) could be used for this purpose. We discovered that nuclear HMGB1 overexpression indicates that death occurred within the previous 12 h, nuclear HMGB1 negativization with high cytoplasmic HMGB1 intensity indicates that death occurred between 12 and 36 h earlier and cytoplasmic HMGB1 negativization indicates that more than 48 h have passed since death. RAGE and Beclin1 levels in the cytoplasm also decreased with time. The latter proteins' negativization might indicate that more than 24 and 36 h, respectively, have passed from the time of death. These indicators might potentially be helpful in forensic practice for determining the PMI using immunohistochemistry.

Parasitic and predatory behavior of Alysia manducator (Hymenoptera: Braconidae) on blow fly larvae feeding on an adult pig carcass in the Western Cape Province of South Africa: preliminary observations and forensic implications.

Wasps are part of the entomofauna associated with vertebrate carrion. They are known to parasitize and prey on specific life stages of insect hosts such as eggs, larvae, pupae, and/or adults associated with vertebrate carrion. However, reports of parasitic behavior of wasps on carrion-associated insect life stages and their possible forensic implications are non-existent in the Western Cape Province of South Africa. This study is part of ongoing research investigating the entomofauna and their pattern of succession on an adult pig carcass in Cape Town, South Africa. During this study, the parasitic wasp Alysia manducator was noted parasitizing and preying on blow fly larvae associated with the decomposing carcass. The arrival of A. manducator coincides with the occurrence of blow fly eggs and/or larvae on the carcass. These wasps were seen in close association with the eggs and larvae of blow flies on various parts of the carcass and some wasps were seen dragging fly larvae attached to their ovipositors away from one part of the carcass to another. Some A. manducator were also observed walking over several larvae on the carcass while exhibiting a stabbing behavior presumably in search of a host for oviposition. We suggest that the observations recorded in this study are of considerable forensic importance as the dragging effect and predatory and stabbing behavior exhibited by A. manducator could potentially disrupt the feeding and development of the fly larvae on the carcass. This could subsequently alter the process of carcass decomposition and/or affect minimum post-mortem interval estimations.

Field validation of post-mortem interval estimation based on insect development. Part 1: Accuracy gains from the laboratory rearing of insect evidence.

There is a consensus that forensic methods must be valid. The high quality of the method may be fully demonstrated only through validation. Unfortunately, there are very few experimental or casework validations of entomological methods for estimating post-mortem interval (PMI). Here, we present the first part of the results from the field validation of minimum PMI (PMImin) estimates based on insect development. From eight pig carcasses (24-46.4 kg) exposed in a forest habitat of Western Poland, one every one or two months, we collected insect evidence with standard entomological techniques. Using weather station temperatures and the thermal summation method, PMImin was estimated based on insect life stages that were reared under controlled laboratory conditions. Through rearing an insect until the next developmental landmark (or until eclosion), its age at the time of collection (and eventually PMImin) may be estimated by subtracting thermal accumulation in the laboratory from the corresponding thermal constant. We hypothesized that rearing insect evidence significantly improves the accuracy of PMImin compared to the estimation based on non-reared insect evidence. The results clearly supported this hypothesis. However, the accuracy significantly increased only in the case of these insects that developed normally during rearing. When their development in the laboratory was prolonged, PMImin was significantly less accurate. For the normal development sample the accuracy improved in all species and life stages. The largest accuracy gains from rearing were recorded for Calliphora vomitoria (Diptera: Calliphoridae) and Stearibia nigriceps (Diptera: Piophilidae). Moreover, when puparia or third instar larvae were reared, gains were larger than in the case of earlier life stages. In conclusion, this study demonstrated that the method of rearing insect evidence to improve the accuracy of PMImin is valid. However, it needs to be used with caution, since substantial part of the evidence may die or slow down their development in the laboratory, which violates assumptions of the method.