Search Results (86)

Background: Accurate estimation of the postmortem interval (PMI) is critical in forensic death investigations. Traditional signs of death—algor mortis, livor mortis, and rigor mortis—are generally reliable only within the first two to three days after death, with their accuracy decreasing as decomposition progresses. This paper presents a systematic review conducted in accordance with PRISMA guidelines, aiming to evaluate and compare current methods for estimating the PMI. Specifically, the study identifies both traditional and modern techniques, analyzes their advantages, limitations, and applicable timeframes, critically synthesizes the literature, and highlights the importance of combining multiple approaches to improve accuracy. Methods: A systematic search was conducted in the PubMed, Scopus, and Web of Science databases, following the PRISMA guidelines. The review included original articles and reviews that evaluated PMI estimation methods (through thanatological signs, entomology, microbial succession, molecular, imaging, and omics approaches). Extracted data included study design, methodology, PMI range, and accuracy information. Out of the 1245 identified records, 50 studies met the inclusion criteria for qualitative synthesis. Results: Emerging methods, such as molecular markers, microbial succession, omics technologies, and advanced imaging show improved accuracy across extended postmortem intervals. RNA degradation methods demonstrated higher accuracy within the first 72 h, while entomology and microbial analysis are more applicable during intermediate and late decomposition stages. Although no single method is universally reliable, combining traditional and modern approaches tailored to case-specific factors improves overall PMI estimation accuracy. Conclusions: This study supports the use of an integrative, multidisciplinary, and evidence-based approach to improve time-since-death estimation. Such a strategy enhances forensic outcomes by enabling more precise PMI estimates in complex or delayed cases, increasing legal reliability, and supporting court-admissible expert testimony based on validated, multi-method protocols. Full article

The pupal stage in necrophagous flies represents the longest and least morphologically distinct phase of development, posing a persistent challenge for accurately estimating postmortem intervals (PMI) in forensic investigations. Here, we present a novel molecular approach to pupal age estimation in Sarcophaga peregrina, a forensically important species, by profiling microRNA (miRNA) expression dynamics. High-throughput sequencing across early, mid, and late pupal stages identified 191 known miRNAs, of which nine exhibited distinct monotonic temporal trends. Six miRNAs (miR-210-3p, miR-285, miR-927-5p, miR-956-3p, miR-92b, and miR-275-5p) were validated by qRT-PCR and demonstrated consistent time-dependent expression patterns. Polynomial regression models revealed a strong correlation between miRNA abundance and developmental age (R² = 0.88–0.99). Functional enrichment analyses of predicted miRNA targets highlighted their roles in key regulatory pathways, including ecdysteroid signaling, hypoxia response, autophagy, and energy metabolism. This study establishes, for the first time, a robust miRNA-based framework for estimating pupal age in forensic entomology, underscoring the potential of miRNAs as temporally precise biomarkers for PMI estimation. Full article

The postmortem interval (PMI), defined as the time elapsed between death and the discovery or examination of the body, is a crucial parameter in forensic science for estimating the time of death. There are many ways to measure the PMI, such as Henssge’s nomogram, which uses rectal temperature measurement; livor mortis; rigor mortis; and forensic entomology. However, these methods are usually affected by various conditions in the surrounding environment. The purpose of the present study was to compare molecular genetics and histological changes in the brain and skeletal muscle tissues of SD rats over increasing periods of time after death. For the PMIs, we considered 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 4 days, 6 days, 8 days, 10 days, 14 days, and 21 days and compared them at 4 °C and 26 °C. Hematoxylin and Eosin (H&E) staining was performed to observe tissue changes. Morphological tissue changes were observed in cells for up to 21 days at 4 °C, and cell destruction was visually confirmed after 14 days at 26 °C. Total RNA (tRNA) was isolated from each tissue sample, and complementary DNA (cDNA) was synthesized. A reverse transcription quantitative PCR (RT-qPCR) SYBR Green assay targeting three types of housekeeping genes, including Gapdh, Sort1, B2m, and 5S rRNA, was performed. The results showed that Gapdh and 5S rRNA were highly stable and could be better RNA targets for estimating the PMI in brain and skeletal muscle tissues. Conversely, Sort1 and B2m showed poor stability and low expression levels. In conclusion, these molecular biomarkers could be used as auxiliary indicators of the PMI in human, depending on the stability of the marker. Full article

Forensic entomotoxicology is a subdiscipline that utilizes necrophagous insects as bioindicators for detecting drugs and toxicants in decomposed remains, particularly in cases where conventional biological matrices are no longer available. Toxic substances can profoundly alter insect development, physiology, and community succession, potentially impacting the accuracy of postmortem interval (PMI) estimation. This review systematically summarizes the effects of various xenobiotics, including pesticides, illicit drugs, sedatives, heavy metals, and antibiotics on larval growth, physiological traits, and gut microbial composition in forensically relevant flies. However, most studies to date have relied primarily on phenotypic observations, with limited insight into underlying molecular mechanisms. Significant interspecies and dose-dependent variability also exists in the absorption, metabolism, and physiological responses to xenobiotics. We highlight recent advances in multi-omics technologies that facilitate the identification of molecular biomarkers associated with xenobiotic exposure, particularly within the insect detoxification system. Key components such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and ATP-binding cassette (ABC) transporters play essential roles in xenobiotic metabolism and insecticide resistance. Additionally, the insect fat body serves as a central hub for detoxification, hormonal regulation, and energy metabolism. It integrates signals related to xenobiotic exposure and modulates larval development, making it a promising model for future mechanistic studies in insect toxicology. Altogether, this review offers a comprehensive and reliable framework for understanding the complex interactions between toxic substance exposure, insect ecology, and decomposition in forensic investigations. Full article

This review presents an in-depth analysis of the synergistic role of molecular biology in advancing forensic entomology. The study discusses how insects associated with decomposing bodies provide critical data for estimating the post-mortem interval (PMI), and how molecular techniques improve species identification and trace analysis. The manuscript examines DNA-based methods such as RAPD, RFLP, and mitochondrial sequencing, along with innovative applications like gene expression profiling and entomotoxicology analysis. Additionally, it presents real case studies illustrating how molecular data from insects can be used not only to estimate PMI but also to identify victims or suspects through human DNA retrieved from insect tissues. These advances confirm the fundamental role of molecular biology in strengthening the reliability and applicability of forensic entomology in legal contexts. Full article

The flesh fly, Wohlfahrtia nuba (Wiedemann) (Diptera: Sarcophagidae), is one of the first necrophagous insects to arrive on a cadaver and is vital for understanding decomposition. Environmental factors, especially temperature, influence insect development, which is crucial for estimating postmortem interval (PMI) in forensic entomology. This study explored how seasonal temperature variations affect the survival and development of W. nuba’s immature stages. The W. nuba colony was reared in the laboratory for four seasons from 3 October 2023 to 30 September 2024. The duration of the larval and pupal phases, the percentage of survival and mortality of the larvae and pupae, the larval growth rate, the percentage of emergence, fecundity, the sex ratio, and the pre-larviposition period were among the many life cycle characteristics that were documented during the study. Research indicates that seasonal changes affect development, shortening the growth period as temperatures rise. Flies raised at an average temperature of 38.3 °C grew faster but experienced higher larval mortality and lower survival rates. The average duration of larval and pupal stages was reduced, with an optimal development temperature of 27.9 °C showing higher survival rates, maximum body weight, and fecundity. The largest mortality rate occurred during winter at an average temperature of 18.5 °C, with males and females showing significant pupal elongation. The findings could help forensic entomologists working on legal investigations to ascertain PMI. Full article

The postmortem interval (PMI) is defined as the time interval between physiological death and the examination of the corpse, playing a critical role in forensic investigations. Traditional PMI estimation methods are often influenced by subjective and environmental factors. Circular RNAs (circRNAs), known for their stability, abundance, and conservation in brain tissue, show promise as biomarkers for PMI estimation. However, research on circRNAs in this context remains limited. This study aimed to develop PMI estimation models using circRNAs across multiple temperatures. By employing semi-quantitative reverse transcription-PCR, circTulp4, circSlc8a1, and circStrn3 were identified as reliable biomarkers for mouse brain tissue. Mathematical models were constructed using the reference genes 28S rRNA, mt-co1, and circCDR1as. At 4 °C, most equations had p-values below 0.05, with the equation using circSlc8a1 as a marker exhibiting the highest goodness of fit. Validation results indicated that the equation using circTulp4 as the reference gene had the highest accuracy. When applying the combined aforementioned three circRNAs, the equation using circCDR1as as the reference gene showed better accuracy. At 25 °C, all equations had R² values greater than 0.86, but most cubic equations had p-values above 0.05. Validation results demonstrated that the circTulp4/mt-co1 equation had the highest accuracy. When applying combined circRNAs, the R² values improved, and long-term PMI estimation was more accurate than short-term PMI estimation. At 35 °C, the linear equations had significantly poorer goodness of fit compared to nonlinear equations, and nonlinear equations exhibited better accuracy than linear equations. When applying the combined aforementioned three circRNAs, the accuracy of the three reference genes was similar, and the accuracy of long-term PMI estimation was consistently higher than that of short-term estimation. For the three-dimensional models, all R² values exceeded 0.75 with p-values significantly below 0.0001. Validation results demonstrated higher accuracy at 25 °C and 35 °C, with superior performance for long-term PMI estimation. In summary, this study constructed PMI estimation models under multiple temperature conditions based on highly expressed circRNAs in mouse brain tissue, highlighting circTulp4, circSlc8a1, and circStrn3 as novel biomarkers. These findings offer a complementary tool for PMI estimation, particularly for long-term PMI estimation. Full article

Background: Accurately determining the post mortem interval (PMI) is critical in forensic investigations to reconstruct the sequence of events leading up to and following death. Traditional methods (e.g., observing livor mortis, rigor mortis, and using temperature-based models) have limitations due to their empirical nature and susceptibility to environmental variables. The objective of this study was to assess the potential of a novel portable device, the Fisic Medimate™ system, for estimating PMI through the analysis of the potassium/sodium (K+/Na+) ratio in vitreous humor. Method: Vitreous humor samples were collected from pigs at various intervals up to 44.5 hours post-mortem. These samples were analyzed using the Fisic Medimate™ system to determine the K+/Na+ ratio. The analysis was conducted at different time points to establish a relationship between the K+/Na+ ratio and PMI. Results: The results indicated a log-linear relationship between the K+/Na+ ratio and PMI for periods up to 31 hours, with normal residuals. This relationship was observed across all samples, suggesting that the method provides reliable and consistent data. Conclusions: This method offers a rapid and portable solution for PMI determination, providing valuable data within minutes directly at the crime scene. While these findings suggest potential for on-site forensic applications, further validation under real-world conditions is required to confirm its broader applicability. Full article

Background: In forensic investigations, the postmortem interval (PMI) is still mainly determined using pathological analysis. There have been many scientific efforts to identify alternative methods of PMI determination, which may be applied to future forensic practices. Methods: Considering the ethical implications and the availability of tissue samples for PMI experiments, we used human blood samples stored at three temperatures to mimic different environmental conditions, testing them over a period of 10 days post-sampling. These samples were biochemically tested for specific blood biomarkers, glucose (Glu) and lactic acid (Lac), to determine their potential as PMI biomarkers. Then, a mixed-effect mathematical model was applied to the data related to time- and temperature-dependent concentration changes of both biomarkers followed by additional computer-simulated models to refine the PMI estimates based on each of the biomarker concentration changes. Results: Herein, we present this alternative method of PMI estimation based on the biochemical testing of blood samples that could potentially be collected at a crime scene using biochemical blood biomarkers Glu and Lac, which are mathematically modelled and refined with time- and temperature concentration changes. Conclusions: While there is still much forensic science required to validate any alternative PMI methods, this study shows that there are other cross-disciplinary methods of PMI determination that warrant further exploration. Full article

Microbiota have emerged as a promising tool for estimating the post-mortem interval (PMI) in forensic investigations. The role of oral and nasal microbiota in cadaver decomposition is crucial; however, their distribution across human cadavers at different PMIs remains underexplored. In this study, we collected 88 swab samples from the oral and nasal cavities of 10 healthy volunteers and 34 human cadavers. Using 16S rRNA gene sequencing, we conducted comprehensive analyses of the alpha diversity, beta diversity, and relative abundance distribution to characterize the microbial communities in both healthy individuals and cadavers at varying PMIs and under different freezing conditions. Random forest models identified Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, and Fusobacteriota as potential PMI-associated biomarkers at the phylum level for both the oral and nasal groups, along with genus-level biomarkers specific to each group. These biomarkers exhibited nonlinear changes over increasing PMI, with turning points observed on days 5, 12, and 22. The random forest inference models demonstrated that oral biomarkers at both the genus and phylum levels achieved the lowest mean absolute error (MAE) values in the training dataset (MAE = 2.16 days) and the testing dataset (MAE = 5.14 days). Additionally, freezing had minimal impact on the overall phylum-level microbial composition, although it did affect the relative abundance of certain phyla. At the genus level, significant differences in microbial biomarkers were observed between frozen and unfrozen cadavers, with the oral group showing greater stability compared to the nasal group. These findings suggest that the influence of freezing should be considered when using genus-level microbial data to estimate PMIs. Overall, our results highlight the potential of oral and nasal microbiota as robust tools for PMI estimation and emphasize the need for further research to refine predictive models and explore the environmental factors shaping microbial dynamics. Full article

Molecular diversity, which reflects variation in species abundance and genetic structure, plays a pivotal role in forensic entomology by enabling the accurate identification of insect evidence through tools such as DNA barcoding. In Pakistan, the absence of trained forensic entomologists and limited research on insect biodiversity hinder the effective use of entomological evidence in criminal investigations. Traditional morphological identification methods are insufficient for resolving complex forensic cases, particularly when dealing with immature insect stages. This highlights the urgent need for molecular approaches, such as DNA barcoding, to enhance species identification and genetic analysis of forensically relevant insects. This study uniquely focuses on evaluating the utility of a 658 bp fragment of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene for identifying dipteran species collected from cadavers in Lahore, Pakistan. The primary goal was to identify forensically relevant insect species, assess their genetic diversity and population structure, and compare these findings with global data to contextualize the results within forensic entomology. Three blow fly species were identified: Chrysomya megacephala (Fabricius, 1794), Chrysomya saffranea (Bigot, 1877), and Chrysomya rufifacies (Macquart, 1843). Low genetic diversity was observed within populations, while significant genetic differentiation among populations was indicated by a high fixation index (FST = 0.83992). These findings suggest unique genetic signatures for blow fly populations in Lahore. This study underscores the importance of molecular tools like DNA barcoding for species identification and highlights the need for further research to establish a comprehensive database of forensically relevant insects in Pakistan, given the limited species diversity and unique genetic profiles observed. By laying the groundwork for future research, this study contributes to advancing forensic entomology in Pakistan by improving species identification, which, when combined with future thermobiological data, can enhance postmortem interval (PMI) estimation and forensic investigations. Full article

Teeth are highly durable and useful in forensic identification and studying the impact of environmental factors could aid forensic investigations. Accurate post-mortem interval (PMI) estimation using dental evidence is critical in legal contexts, it requires further exploration. Aims: This study included a scoping review investigating macroscopic and microscopic changes in teeth in various simulated environments (Part 1) and an experimental study assessing changes in teeth and restorations exposed to distilled water, saline water, acidic soil, and alkaline soil (Part 2). Methods: The scoping review analysed publications from five databases using keywords such as ‘Teeth’, ‘Dental’, ‘Water’, ‘Soil’, ‘Acid’, and ‘Forensic.’ The experimental study involved 40 human teeth photographed before and after a 90-day exposure period to record shade variations and macroscopic changes. Results: Part 1: Twenty-six relevant articles from 10 countries (1987–2022) were reviewed, with most focusing on human teeth (77%), unrestored teeth (54%), macroscopic changes (46%), and high-temperature environments (53%). Part 2: Teeth in distilled water (G1) showed no shade variation. In saline water (G2), 60% of teeth decreased in shade. In acidic soil (G3), 40% showed an increased shade, while 50% showed a decreased shade. In alkaline soil (G4), 70% of teeth showed an increased shade. Restorations exhibited minimal changes across environments. Conclusions: Studies on the macroscopic changes because of high temperature on teeth and dental restorative material are popular. Teeth exposed to alkaline and acidic soil showed the most changes in the structure. Full article

Objectives: Determination of the postmortem interval (PMI) remains a critical challenge in forensic science. Intervertebral discs, due to their structural resilience, hold promise as a reliable tissue for PMI estimation; however, studies focusing on their forensic applicability remain limited. This study aimed to evaluate progressive histopathological changes in intervertebral discs at specific postmortem intervals and assess their forensic applicability. Materials and Methods: A total of 48 rats were divided into six groups: control (0 h), 7-day, 15-day, 30-day, 60-day, and 90-day postmortem intervals. Intervertebral disc samples were stained with hematoxylin–eosin and trichrome, and histopathological parameters such as homogenization, eosinophilia, dissociation, nuclear alterations (pyknosis and karyolysis), and collagen fragmentation were analyzed. Results: Statistically significant changes were observed across postmortem intervals (p < 0.001). Homogenization progressed from mild changes at 7 days to prominent levels by 90 days. Eosinophilia and dissociation between the epithelium and connective tissue also increased significantly over time (p < 0.001). Collagen fragmentation, initially minimal, became severe at the 90-day interval. The observed changes demonstrated a clear, time-dependent progression strongly correlating with the PMI. Conclusions: Our findings suggest that histopathological changes in intervertebral discs follow a consistent and time-dependent pattern, making them a potential forensic marker for PMI estimation. This has important implications for forensic science, as it offers an alternative tissue type that is less susceptible to early decomposition compared to soft tissues. These results suggest that the intervertebral disc is a promising tissue for PMI estimation, offering a complementary approach to existing forensic methods. Full article

Estimating the minimum post-mortem interval (PMI-min) is crucial in criminal investigations for identifying victims and the circumstances surrounding their death. Traditional post-mortem indicators are reliable only within the first 48–72 h post-mortem. This study explored forensic entomology as an alternative method for PMI estimation, focusing on three cases in Busan, South Korea. Forensic cases involving insect specimens collected from bodies found in Busan from 2022 were examined. Personal and photographic data were documented, and insect specimens were collected, preserved, and identified based on both their morphological characteristics and DNA sequences. To enhance the estimation accuracy, corrected death scene temperatures were calculated using an electronic thermo-hygrometer and meteorological data, applying both quadratic regression and the Support Vector Machine (SVM) model. The PMI-min was estimated using growth models and developmental data from established studies. Lucilia sericata (Meigen) at different life-cycle stages were discovered in all of the cases, whereas Chrysomya megacephala (Fabricius) was found in only two out of the three cases. In each case, the estimated time of death based on the necrophagous flies differed from the deceased’s last known activity by approximately one–two days. These discrepancies may arise from the pre-colonization interval (PCI), a critical but often overlooked factor for accurate PMI-min estimation. Additional factors, including weather conditions, oviposition timing, mixed fly populations, and maggot-generated heat, further contribute to the uncertainty of PMI-min estimates. Future research should integrate these variables and employ advanced technologies such as machine learning to improve the accuracy of these estimates. Full article

Cuticular hydrocarbons (CHCs) are long-chain lipids found on the exoskeletons of insects, serving primarily as a protective barrier against water loss and environmental factors. In the last few decades, the qualitative and quantitative analysis of CHCs, particularly in blow flies, has emerged as a valuable tool in forensic entomology, offering promising potential for species identification and age estimation of forensically important insects. This review examines the current application of CHC analysis in forensic investigations and highlights the significant advancements in the field over the past few years. Studies have demonstrated that CHC profiles vary with insect development, and while intra-species variability exists due to factors such as age, sex, geographical location, and environmental conditions, these variations can be harnessed to refine post-mortem interval (PMI) estimations and improve the accuracy of forensic entomological evidence. Notably, CHC analysis can also aid in distinguishing between multiple generations of insects on a body, providing insights into post-mortem body movement and aiding in the interpretation of PMI in complex cases. Furthermore, recent studies have investigated the variability and degradation of CHCs over time, revealing how environmental factors—such as temperature, humidity, UV light exposure, and toxicological substances—affect CHC composition, providing valuable insights for forensic investigations. Despite the promise of CHC profiling, several challenges remain, and this review also aims to highlight future research directions to enhance the reliability of this technique in forensic casework. Full article