Forensic Sci., Volume 5, Issue 2 (June 2025) – 12 articles

Background: The identification of body fluids collected from crime scenes is crucial for determining the type and nature of assaults and for advancing the resolution of crimes. Objectives: The primary aim of this study was to investigate tissue-specific DNA methylation markers that can effectively distinguish buccal samples from blood, semen, and vaginal epithelial tissue. Methods: We screened various markers and selected four genomic locations for further analysis. Genomic DNA was extracted from tissue samples, followed by bisulfite conversion, locus-specific polymerase chain reaction (PCR) amplification, and pyrosequencing. Results: Four loci—cg-9652652, cg-11536474, cg-3867465, and cg-10122865—along with several adjacent CpG sites, were found to be hypermethylated in buccal samples compared to other tissue types. The difference in DNA methylation of buccal samples was statistically significant (p < 0.0001) compared to other tissues, indicating the potential usefulness of these loci for forensic tissue identification. Two additional studies were conducted: (a) a species specificity study and (b) a mixture study involving two different tissue types. The species specificity study showed that the primers used in the assay were specific to primates and humans. They did not amplify five non-primate samples, while the two primate samples—chimpanzee and rhesus—provided usable methylation data. The mixture study involved DNA from two different tissues—buccal samples and semen—combined in varying proportions. The results showed a decrease in the overall percentage of DNA methylation at the locus cg-9652652 as well as five adjacent CpG sites when the amount of buccal cell DNA in the mixture was reduced. Conclusion: The specificity of the primers and the significant differences in percent DNA methylation between buccal cells and other tissues make these markers excellent candidates for forensic tissue identification. Full article

The calcaneus is very useful in archaeological contexts where human remains may be commingled. When DNA is impossible and the context is commingled, non-metric traits of the calcaneus may be used to identify phenotypical differences between human remains. Background/Objectives: We compared the prevalence of several calcaneal non-metric traits of pre-Hispanic and modern inhabitants of the Canary islands in order to (1) test the skeletal variation between pre-Hispanic and modern (17–18th century) Canarian samples and (2) gather information on biological and lifestyle differences between the samples using the calcaneal bone. Methods: We used a total of 364 calcanei (164 modern Canarian [72 left and 92 right] and 200 pre-Hispanic Canarian [92 left and 108 right] calcanei) and available non-metric traits to show differences between modern and pre-Hispanic Canarian population samples. Results: Our results highlight that some particular activity traits were common among the pre-Hispanic sample, while other traits, such as articular facet type Ib and the medial root of the inferior extensor retinaculum, show similar frequencies between pre-Hispanic and modern samples, which may indicate a genetic proponent influencing these similarities. Conclusions: Our results suggest that at least two traits, facet type Ib and the medial root of the inferior extensor retinaculum, may be influenced by genetics due to the persistence in modern samples despite the change of lifestyle between pre-Hispanic and modern Canarians. Full article

(1) Background: Scavenger activity can pose significant challenges in forensic investigations. When examining damage on a victim, forensic investigators must first determine whether the damage resulted from human actions or animal scavenging. Accordingly, knowledge of the scavenger populations inhabiting a specific region and their scavenging behaviors is essential for accurately reconstructing the environmental context of the remains and interpreting associated taphonomic evidence in forensic investigations. (2) Methods: This study investigates the scavenging behavior of bobcats (Lynx rufus), a species native to North America, under controlled experimental conditions. The authors placed the carcasses of seven adult white-tailed deer in an outdoor field in Murfreesboro, Tennessee, and systematically documented the scavenging activities. (3) Results: Bobcats were observed to primarily scavenge soft tissue from the torso, focusing on musculature while generally avoiding bony regions. When unsatisfied with the torso, they shifted attention to the upper limbs, with minimal interest shown in the head or neck. Bobcats also exhibited a tendency to feed in a single area for extended periods, leading to concentrated damage in specific locations on the remains. (4) Conclusions: The findings demonstrate that bobcats can significantly influence the postmortem conditions of remains, producing characteristic patterns of modification. Recognizing these patterns is essential for forensic investigators and examiners when interpreting scavenger-related alterations in forensic contexts. Full article

The decision to remove a bullet from a gunshot victim depends on its location and associated medical risks, with surgical extraction often not indicated. Radiological imaging plays a vital role in assessing gunshot wounds and locating bullets, and it is essential in both clinical and forensic contexts. This narrative review examines the use of computed tomography (CT) for virtual bullet analysis, providing insights into shape, design, fragmentation, and material composition. Traditional 2D X-ray imaging, though commonly used, has limitations in accurately assessing caliber and position due to magnification and its 2D nature. In contrast, CT scans generate 3D reconstructions for detailed and precise examination, overcoming challenges such as metal artifacts with techniques such as extended Hounsfield unit (HU) reconstructions. These methods enhance the visualization of metal objects, allowing for better analyses of lodged bullets. Dual-energy CT further differentiates materials, such as lead and copper, using HU value differences at two energy levels. These advancements enable the virtual classification, shape analysis, and material identification of bullets in forensic investigations, even when the bullet remains in the body. As CT technology progresses, its forensic applications are expected to improve, providing more accurate and comprehensive differentiations of bullet types in future cases. Full article

Lithium is used as a medication in the treatment of bipolar disorder. Lithium has a narrow therapeutic index, and fatal intoxications have been described. The therapeutic drug monitoring of lithium is routinely performed in serum. Serum is commonly unavailable in forensic postmortem analysis, where whole blood is the matrix of choice. In this study, an inductively coupled plasma mass spectrometry (ICP-MS) method was developed and validated for the quantification of lithium in postmortem whole blood. Sample preparation consisted of a 100-fold dilution with acid and required only 40 µL of blood. Carry-over was deemed appropriately reduced with a rinse solution containing 5% hydrochloric acid. A nebulizer gas flow rate of 1.15 L/min showed a sufficient improvement of lithium sensitivity while simultaneously minimizing the background. Germanium was determined to be the most optimal internal standard. The method was validated in terms of linearity, accuracy, precision, and lower limit of quantification. Linearity was demonstrated within the analytical measurement range of 0.10–1.5 mmol/L. The method showed acceptable precision and accuracy, with a total coefficient of a variation ≤2.3% and accuracies ranging from 105 to 108% at all concentrations in the quality control samples. The final method was applied to postmortem blood from 103 consecutive autopsy cases and demonstrated robustness by low intermediate precision and high and consistent recovery of the internal standard. Full article

Background: Chemical odor profiling within forensic entomology is an emerging tool given its potential for species identification and larval aging and its ability to identify decomposition stages. A volatile analysis of larval masses across species of distinctive developmental stages was carried out with extraction techniques to identify odor signatures. However, it is unknown how larval sample handling (i.e., live samples in research vs. hot-water-killed samples in casework) affects odor signatures or the possibility of obtaining relevant volatiles from a single larva. Method: This study utilized solid-phase microextraction (SPME) with gas chromatography–mass spectrometry (GC/MS) for the analysis of odor volatiles emanating from single larval samples of Cochliomyia macellaria. Fifty (50) larvae (25 live; 25 boiled) were analyzed. Results: The SPME-GC/MS method allowed for odor volatile detection from a single maggot regardless of the sample handling group. The main compounds identified across both groups included those previously reported as emanating from larvae and decomposition substrates. When comparing treatments, the boiled larval samples had a 6-fold decrease in compound abundance compared to the live samples. The identified odor volatiles observed in the hot-water-killed treatment group included indole, p-cresol, and phenol. Conclusions: These results suggest that the handling technique impacts odor detection. Additionally, the heterocyclic aromatics and alcohols identified in the boiled samples are potentially odor markers of a higher intrinsic nature to the maggot rather than a cross-transfer from the decomposition substrate given their survival post elevated temperature treatment. This work shows the plausibility of carrying out an odor analysis of a single maggot following both common research and casework handling practices. Full article

Background: The COVID-19 pandemic significantly disrupted forensic science, exposing vulnerabilities and introducing unprecedented challenges. Five years later, its impact persists, necessitating ongoing adaptations in forensic practice. This study examines key transformations, persistent issues, and emerging challenges in forensic science post-pandemic. Methods: A critical analysis of forensic science’s response to the pandemic was conducted, focusing on operational disruptions, methodological advancements, educational shifts, and technological integration. Results: Forensic operations faced delays due to case backlogs, restricted in-person work, and postponed court proceedings. Forensic pathology evolved with increased reliance on molecular autopsy techniques to clarify COVID-19-related deaths. Educational methods shifted toward virtual learning, prompting discussions on standardized digital training. Additionally, artificial intelligence and automation gained prominence in forensic investigations, enhancing crime scene analysis and predictive modeling. Discussion: While forensic science demonstrated adaptability, challenges remain in international collaboration, resource distribution, and professional training. The pandemic accelerated technological integration but also raised ethical and procedural concerns, particularly regarding AI applications in legal contexts. Virtual learning innovations necessitate further development to ensure competency in forensic training. Conclusions: Forensic science continues to evolve in response to post-pandemic realities. Addressing gaps in cooperation, technology implementation, and training will be crucial to strengthening the field. By assessing these changes, this study underscores forensic science’s resilience and adaptability, offering insights into its future trajectory amid ongoing challenges. 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

Contemporary human preservation technologies aim to preserve the structure of the body—and especially the brain—after legal death. Although it has not yet been demonstrated, some believe that this may act as a bridge to future medical technologies that could allow for the recovery of life, if this ever becomes feasible and humane. However, current death investigation practices impose two significant obstacles to high-quality preservation. The first and most common is ischemic delay—the period between legal death and initiation of preservation while the death is investigated, during which brain tissue progressively degrades. The second is involuntary autopsy, where tissue disruption can severely compromise preservation quality. Through analysis of legal precedents and the ethical principles of bodily autonomy, non-discrimination, and potential preservation of life, we argue that individuals should be allowed a way to prevent both ischemic delay and autopsy from affecting the quality of their preservation. We explore potential avenues to implement a right to immediate preservation, including administrative, legislative, and judicial approaches, aimed at balancing this right with the societal interest in performing death investigations. This narrative review has implications for individual autonomy, public policy, and the legal framework surrounding death investigation in the context of emerging preservation technologies. Full article

Background: The main objective of this study was to carry out an online questionnaire in order to assess dentists’ knowledge and perception of dental materials submitted to high temperatures, helping to clarify and raise awareness of possible gaps in the area of forensic dentistry, especially in human identification. Results: Out of a total of 272 respondents, there was a greater influx of responses from countries such as Portugal and Brazil, the majority being female (66.3%), with an average age of 39 years. Most dentists consider complete clinical records to be extremely important. In addition, professionals with less experience provide fewer models in releasing their clinical records. In the case of dental materials, dentists consider composite resin and glass ionomers to be more susceptible to changes in shape and colour, while amalgam is considered the most stable, followed by ceramic and metal-ceramic crowns. On the other hand, dentin is considered more susceptible to colour changes than enamel. Enamel is seen by most professionals as resistant, undergoing few changes in shape at high temperatures. Conclusions: The opinion of the surveyed dentists on this subject is broadly in line with the existing literature. The only observed differences were related to exposure time and enamel resistance, since they assume that enamel is more resistant, and dentin is more vulnerable. Full article