Estimating the post-mortem interval (PMI) is an essential step in forensic investigations, particularly those involving homicides and unwitnessed deaths. However, traditional methods occasionally yield inconsistent estimates. Histological and molecular techniques are considered crucial in forensic pathology and are frequently employed to estimate the time interval of death. The gingiva is an oral mucosal tissue used to estimate PMI. This review aimed to examine the potential of histological methods to determine PMI using oral mucosal tissue, namely the gingiva, and to investigate changes that occur in oral mucosal tissue at different time intervals when compared with those in normal tissues. The oral mucosa comprises layers of stratified squamous epithelium and connective tissue. Similar to other body tissues, changes are known to occur in the gingiva after death, and these cellular and tissue changes should also be considered. Alterations in the gingiva include homogenisation, karyorrhexis, pyknosis, karyolysis, chromatin clumping, eosinophilia, collagen fibre degradation, and the loss of tissue architecture. Reviews collating the results of original trials have consistently reported how the oral mucosa is altered by autolysis and how such changes can be observed in histological tissue morphology after death. Histology is an acceptably accurate technique for estimating PMIs.

Postmortem interval (PMI) in legal medicine is extremely important for both criminal and civil cases, and several sorts of techniques have been recommended. This systematic review solely focuses on approaches linked to RNA analysis, instead of including all proposed methods for determining the PMI. The term PMI will be used in this review to indicate the time between a person\'s death and the postmortem examination of the body. We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Guidelines when conducting this systematic review. The majority of studies on various tissues at various time intervals at various temperatures are non-human, and just a small number are on humans. The results are then provided using various statistical approaches. To calculate the PMI, post-mortem RNA degradation was examined using several tissues. The result so obtained had an opposite polarity. While some studies show that RNA stability in various tissues remained constant for several days after death, the other group of studies showed evident RNA degradation over time post-mortem, which was significantly influenced by temperature and other agonal factors. These factors have an impact on the multi-parametric mathematical model of ante and post-mortem factors on RNA degradation, as well as its applicability and feasibility. The estimation of PMI using RNA degradation can prove to be highly objective and efficient after controlling for the various factors and challenges that pose the estimation of RNA in forensic samples difficult.

The K+ and hypoxanthine (Hx) concentrations of the vitreous humour (VH) rise gradually after death, providing a means of estimating the post-mortem interval (PMI). The correlation between these analytes and the PMI is good since the vitreous chamber is partially isolated from autolytic events occurring elsewhere; the [K +] and [Hx] recorded is thus the result of changes within the eye. The present work provides a systematic review, following PRISMA recommendations, of 36 articles (3 reviews and 33 retrospective cohort studies) discussing the many procedures and regression models that have been developed for improving PMI estimates involving VH analytes. The results of a descriptive study are also provided, highlighting the causes and distribution of mortality as registered in medico-legal autopsies performed in 2019 in Galicia (northwestern Spain), and revealing the use of these PMI estimation methods in real forensic practice. Great heterogeneity was detected in the collection of VH samples, the treatments to which they were subjected before examination, and in their conservation and analysis. A lack of reproducibility in the analytical methods employed to estimate [K +] and [Hx] was noted, as well as an absence of external validation for most of the regression formulae used to determine the PMI from analyte values. The use of methods based on high-performance liquid chromatography, focal electrophoresis, or thermogravimetric/chemometric procedures might solve the problems encountered with traditional analytical techniques, offering reliable results more quickly and effectively (even when samples are contaminated). This study recommends using flexible multiple regression models that combine physical and chemical variables, and that population databases be constructed so that models can be properly validated.

The identification of a reliable and accurate post-mortem interval (PMI) is a major challenge in the field of forensic sciences and criminal investigation. Several laboratory techniques have recently been developed that offer a better contribution to the estimation of PMI, in addition to the traditional physical or physico-chemical (body cooling, lividity, radiocarbon dating, rigor mortis), chemical (autolysis), microbiological (putrefaction), entomological, as well as botanical parameters. Molecular biology (degradation pattern of macromolecules such as proteins, DNA, RNA), biochemical analysis of biological fluids (such as blood, cerebrospinal fluid, and vitreous humor), and immunohistochemistry are some of the most recent technological innovations. A systematic review of the literature was performed with the aim of presenting an up-to-date overview on the correlation between the immunohistochemical (IHC) expression of specific antigenic markers at different PMIs. The systematic review was performed according to PRISMA guidelines. Scopus and PubMed were used as search engines from January 1, 1998 to March 1, 2022 to evaluate the effectiveness of immunohistochemistry in estimating PMI. The following keywords were used: (immunohistochemical) OR (immunohistochemistry) AND (time since death) OR (post-mortem interval) OR (PMI). A total of 6571 articles were collected. Ultimately, 16 studies were included in this review. The results of this systematic review highlighted that IHC techniques, in association with traditional methods, add, in Bayesian terms, additional information to define a more accurate time of death and PMI. However, current IHC results are numerically limited and more data and studies are desirable in the near future.

The main aim of this study was to investigate the post-mortem proteolytic degradation process of selected tissue antigens and correlate it to the post-mortem interval. During the autopsy of 12 cadavers (time interval ranging 1 day-2 years after death) samples of skin, liver, kidney, and spleen were collected. All samples were formalin-fixed and paraffin-embedded. Four µm paraffin sections were used for hematoxylin-eosin staining and immunohistochemical analysis (Ki67, Vimentin, Pan cytokeratin, and CD20). Data reported here show that immunohistochemical reactivity preservation was related to the characteristics of the tissues. In particular, the most resistant tissue was the skin, where the autolysis phenomena were not appreciable before 5 days. On the contrary, the liver and the spleen underwent early autolysis, while the kidney displayed an early autolysis of the tubules and a late one of the glomeruli. As concerns specific antigens, immunoreactivity was lost earliest for nuclear antigens as compared to cytoplasmic ones. In conclusion, our results demonstrate that immunohistochemical detection of specific antigens may be useful in estimating the post-mortem interval, especially when we need to know whether the post-mortem interval is a few days or more than 7-10 days.

Estimating the post-mortem interval (PMI) is a very complex issue due to numerous variables that may affect the calculation. Several authors have investigated the quantitative and qualitative variations of protein expression on post-mortem biological samples in certain time intervals, both in animals and in humans. However, the literature data are very numerous and often inhomogeneous, with different models, tissues and proteins evaluated, such that the practical application of these methods is limited to date. The aim of this paper was to offer an organic view of the state of the art about post-mortem protein alterations for the calculation of PMI through the analysis of the various experimental models proposed. The purpose was to investigate the validity of some proteins as \"molecular clocks\" candidates, focusing on the evidence obtained in the early, intermediate and late post-mortem interval. This study demonstrates how the study of post-mortem protein alterations may be useful for estimating the PMI, although there are still technical limits, especially in the experimental models performed on humans. We suggest a protocol to homogenize the study of future experimental models, with a view to the next concrete application of these methods also at the crime scene.

Currently, forensic research is multidisciplinary with new methods and parameters useful to define the cause and time of death as well as survival/agony times. The identification of biochemical markers able to estimate agonal period has been studied by many forensic researchers. It is known that the estimation of agonal time in different types of death is not always easy, hence our interest in literature\'s data. The studies analyzed in this review confirm the important role of thanatobiochemistry for the estimation of survival times. Regardless of the death cause, the survival/agony time between the primary event and death influences markers concentrations in biological samples (e.g., blood, urine, cerebrospinal fluid). Different biomarkers can be used for qualitative evaluations in deaths with short and long agony (e.g., C-reactive protein, ferritin, GFAP, etc.). Instead, the quantitative interpretation showed limits due to the lack of reference cut-offs. Thanatobiochemistry is a useful tool to confirm what emerged from autopsies findings (macroscopic and histological analysis), but further studies are desirable to confirm the evidence emerging from our review of the literature.

Estimating the time of death remains the most challenging question in forensic medicine, because post-mortem interval (PMI) estimation can be a remarkably difficult goal to achieve. The aim of this review is to analyze the potential of microRNAs (miRNAs) to evaluate PMI. MiRNAs have been studied as hallmarks and biomarkers in several pathologies and have also showed interesting applications in forensic science, such as high sensible biomarkers in body fluid and tissue, for wound age determination and PMI evaluation due to their low molecular weight and tissue-specific expression. The present systematic review was carried out according to the Preferred Reporting Items for Systematic Review (PRISMA) standards. We performed an electronic search of PubMed, Science Direct Scopus, and Excerpta Medica Database (EMBASE) from the inception of these databases to 12 August 2020. The search terms were (\"PMI miRNA\" or \"PMI micro RNA\") and (\"miRNA\" and \"time of death\") in the title, abstract and keywords. Through analysis of scientific literature regarding forensic uses of miRNAs, has emerged that the intrinsic characteristics of such molecules, and their subsequent resistance to degradation, make them suitable as endogenous markers in order to determine PMI. However, further and larger studies with human samples and standardized protocols are still needed.

The determination of the post-mortal interval (PMI) is an extremely discussed topic in the literature and of deep forensic interest, for which various types of methods have been proposed. The aim of the manuscript is to provide a review of the studies on the post-mortem DNA degradation used for estimating PMI. This review has been performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and the PRISMA Guidelines. Several analytical techniques have been proposed to analyse the post-mortem DNA degradation in order to use it to estimate the PMI. Studies focused mainly on animal models and on particular tissues. The results have been mixed: while on the one hand literature data in this field have confirmed that in the post-mortem several degradation processes involve nucleic acids, on the other hand some fundamental aspects are still little explored: the influence of ante and post-mortem factors on DNA degradation, the feasibility and applicability of a multiparametric mathematical model that takes into account DNA degradation and the definition of one or more target organs in order to standardize the results on human cases under standard conditions.

Post-mortem interval (PMI) determination is one of the most important issues in forensic sciences. In the past, forensic scientists provided different approaches (physical, chemical, and entomological) for the estimation of PMI without success.However, advances in molecular biology over the last two decades have allowed us to assess the time-dependent degradation of biological markers (e.g., proteins, DNA, and RNA). Thus, the aim of the manuscript is to provide a review of the recent progress in the estimation of PMI using molecular biology methods, mainly focusing on the potential usefulness of RNA markers. To this end, 29 studies have been systematically reviewed, each one chosen according to specific inclusion/exclusion criteria. The selected studies evaluated the contribution of endogenous reference genes in different biological samples in order to determine the PMI based on post-mortem RNA degradation as a function of other influencing factors such as time, cause of death, and environmental conditions.