Estimating the postmortem interval (PMI) is one of the major tasks and a continuous challenge in forensic pathology. It is often an exclusion process of available methods, which ultimately can lead to an unsatisfactory outcome due to poor reliability. This problem is most acute in the late PMI, when decomposition proceeds and some methods (such as rigor, livor, and algor mortis) are no longer applicable. Several methods, such as forensic entomology, skeletal muscle protein degradation, and the study of body decomposition by application of a morphological scoring, are expected to provide further information; however, all have certain limitations and weaknesses. Availability of a tool-box of methods allows a case-specific selection of the most appropriate one(s), or eventually provides improvements in the overall accuracy and precision of the PMI estimation by merging and combining methods. To investigate practical (field) application, eventual interferences, and/or synergetic effects, as well as the robustness of these methods towards specific influencing factors, a field study was conducted, using eight pig cadavers of different body weights and physical coverage, left to decompose under natural conditions for 16 days. Morphological changes during decomposition were assessed using the total body score (TBS), muscle samples were collected to analyze protein degradation, and insect colonization was evaluated. The results reveal strengths and current limitations of all tested methods, as well as promising synergistic effects, and thus, provide a baseline for targeted future research.

Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.

Objectives: We provide a systematic review of the literature to evaluate the current research status of protein degradation-based postmortem interval (PMI) estimation. Special attention is paid to the applicability of the proposed approaches/methods in forensic routine practice. Method: A systematic review of the literature on protein degradation in tissues and organs of animals and humans was conducted. Therefore, we searched the scientific databases Pubmed and Ovid for publications until December 2019. Additional searches were performed in Google Scholar and the reference lists of eligible articles. Results: A total of 36 studies were included. This enabled us to consider the degradation pattern of over 130 proteins from 11 different tissues, studied with different methods including well-established and modern approaches. Although comparison between studies is complicated by the heterogeneity of study designs, tissue types, methods, proteins and outcome measurement, there is clear evidence for a high explanatory power of protein degradation analysis in forensic PMI analysis. Conclusions: Although only few approaches have yet exceeded a basic research level, the current research status provides strong evidence in favor of the applicability of a protein degradation-based PMI estimation method in routine forensic practice. Further targeted research effort towards specific aims (also addressing influencing factors and exclusion criteria), especially in human tissue will be required to obtain a robust, reliable laboratory protocol, and collect sufficient data to develop accurate multifactorial mathematical decomposition models.

The assessment of postmortem degradation of skeletal muscle proteins has emerged as a novel approach to estimate the time since death in the early to mid-postmortem phase (approximately 24 h postmortem (hpm) to 120 hpm). Current protein-based methods are limited to a small number of skeletal muscle proteins, shown to undergo proteolysis after death. In this study, we investigated the usability of a target-based and unbiased system-wide protein analysis to gain further insights into systemic postmortem protein alterations and to identify additional markers for postmortem interval (PMI) delimitation. We performed proteomic profiling to globally analyze postmortem alterations of the rat and mouse skeletal muscle proteome at defined time points (0, 24, 48, 72, and 96 hpm), harnessing a mass spectrometry-based quantitative proteomics approach. Hierarchical clustering analysis for a total of 579 (rat) and 896 (mouse) quantified proteins revealed differentially expressed proteins during the investigated postmortem period. We further focused on two selected proteins (eEF1A2 and GAPDH), which were shown to consistently degrade postmortem in both rat and mouse, suggesting conserved intra- and interspecies degradation behavior, and thus preserved association with the PMI and possible transferability to humans. In turn, we validated the usefulness of these new markers by classical Western blot experiments in a rat model and in human autopsy cases. Our results demonstrate the feasibility of mass spectrometry–based analysis to discover novel protein markers for PMI estimation and show that the proteins eEF1A2 and GAPDH appear to be valuable markers for PMI estimation in humans.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.

Awareness of postmortem degradation processes in a human body is fundamental to develop methods for forensic time since death estimation (TDE). Currently, applied approaches are all more or less limited to certain postmortem phases, or have restrictions on behalf of circumstances of death. Novel techniques, however, rarely exceed basic research phases due to various reasons. We report the first application of a novel method, based on decay of muscle proteins, in a recent case of murder-suicide, where other TDE methods failed to obtain data. We detected considerably different protein degradation profiles in both individuals involved and compared the data to our presently available database. We obtained statistical evidence for un-simultaneous death and therefore received valuable information to trace the progression of events based on protein degradation. Although we could not sensibly convert the data to respective times of death, this case highlights the potential for future application and elucidates the necessary further steps to develop a viable TDE method.