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Keywords = forensic molecular pathology

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19 pages, 3064 KiB  
Article
HR-pQCT and 3D Printing for Forensic and Orthopaedic Analysis of Gunshot-Induced Bone Damage
by Richard Andreas Lindtner, Lukas Kampik, Werner Schmölz, Mateus Enzenberg, David Putzer, Rohit Arora, Bettina Zelger, Claudia Wöss, Gerald Degenhart, Christian Kremser, Michaela Lackner, Anton Kasper Pallua, Michael Schirmer and Johannes Dominikus Pallua
Biomedicines 2025, 13(7), 1742; https://doi.org/10.3390/biomedicines13071742 - 16 Jul 2025
Viewed by 215
Abstract
Background/Objectives: Recent breakthroughs in three-dimensional (3D) printing and high-resolution imaging have opened up new possibilities in personalized medicine, surgical planning, and forensic reconstruction. This study breaks new ground by evaluating the integration of high-resolution peripheral quantitative computed tomography (HR-pQCT) with multimodal imaging and [...] Read more.
Background/Objectives: Recent breakthroughs in three-dimensional (3D) printing and high-resolution imaging have opened up new possibilities in personalized medicine, surgical planning, and forensic reconstruction. This study breaks new ground by evaluating the integration of high-resolution peripheral quantitative computed tomography (HR-pQCT) with multimodal imaging and additive manufacturing to assess a chronic, infected gunshot injury in the knee joint of a red deer. This unique approach serves as a translational model for complex skeletal trauma. Methods: Multimodal imaging—including clinical CT, MRI, and HR-pQCT—was used to characterise the extent of osseous and soft tissue damage. Histopathological and molecular analyses were performed to confirm the infectious agent. HR-pQCT datasets were segmented and processed for 3D printing using PolyJet, stereolithography (SLA), and fused deposition modelling (FDM). Printed models were quantitatively benchmarked through 3D surface deviation analysis. Results: Imaging revealed comminuted fractures, cortical and trabecular degradation, and soft tissue involvement, consistent with chronic osteomyelitis. Sphingomonas sp., a bacterium that forms biofilms, was identified as the pathogen. Among the printing methods, PolyJet and SLA demonstrated the highest anatomical accuracy, whereas FDM exhibited greater geometric deviation. Conclusions: HR-pQCT-guided 3D printing provides a powerful tool for the anatomical visualisation and quantitative assessment of complex bone pathology. This approach not only enhances diagnostic precision but also supports applications in surgical rehearsal and forensic analysis. It illustrates the potential of digital imaging and additive manufacturing to advance orthopaedic and trauma care, inspiring future research and applications in the field. Full article
(This article belongs to the Section Biomedical Engineering and Materials)
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18 pages, 1130 KiB  
Review
Five Years After the COVID-19 Pandemic: Old Problems and New Challenges in Forensic Pathology
by Mario Chisari, Martina Francaviglia, Sabrina Franco, Gianpietro Volonnino, Raffaella Rinaldi, Nicola Di Fazio and Lucio Di Mauro
Forensic Sci. 2025, 5(2), 20; https://doi.org/10.3390/forensicsci5020020 - 2 May 2025
Viewed by 716
Abstract
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 [...] Read more.
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
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8 pages, 201 KiB  
Article
Validity of the Rapid Nasopharyngeal Antigen Swab for the Detection of SARS-CoV-2 on Cadavers
by Isabella Caristo, Rosario Barranco, Sara Lo Pinto and Francesco Ventura
Forensic Sci. 2025, 5(1), 6; https://doi.org/10.3390/forensicsci5010006 - 4 Feb 2025
Viewed by 716
Abstract
Background: SARS-CoV-2 is classified as a class 3 biological agent; therefore, autopsies on positive subjects must be performed in BSL 3 sectorial rooms. However, many centers lacking such facilities perform molecular nasopharyngeal swabs for SARS-CoV-2 on corpses before autopsy. This approach, though, is [...] Read more.
Background: SARS-CoV-2 is classified as a class 3 biological agent; therefore, autopsies on positive subjects must be performed in BSL 3 sectorial rooms. However, many centers lacking such facilities perform molecular nasopharyngeal swabs for SARS-CoV-2 on corpses before autopsy. This approach, though, is marked by prolonged reporting times and extremely high costs. This study aims to compare the results of molecular swabs (RT-PCR) with rapid antigen swabs (RAT) in order to assess if RAT can serve as the sole test for determining corpse positivity or negativity. Methods: Sixty corpses with positive molecular nasopharyngeal swabs for SARS-CoV-2, performed either ante-mortem or post-mortem, were selected. Afterward, they underwent rapid antigen swabs within 0 to 11 days after the last molecular exam. Results: Out of 60 corpses with positive molecular swabs, 52 antigen swabs were positive (86.67%), and 8 were negative (13.33%), indicating a sensitivity of 86.66% and specificity of 100%. Conclusions: Considering the sensitivity and specificity values observed in this study, RAT could be used as the primary investigation on corpses, especially in centers that lack BSL 3 sectorial rooms. Molecular swabs could then serve as a secondary test for subjects negative on RAT. Full article
17 pages, 593 KiB  
Review
The Role of GFAP in Post-Mortem Analysis of Traumatic Brain Injury: A Systematic Review
by Matteo Antonio Sacco, Saverio Gualtieri, Alessandro Pasquale Tarallo, Maria Cristina Verrina, Jasmine Calafiore, Aurora Princi, Stefano Lombardo, Francesco Ranno, Alessandro Di Cello, Santo Gratteri and Isabella Aquila
Int. J. Mol. Sci. 2025, 26(1), 185; https://doi.org/10.3390/ijms26010185 - 28 Dec 2024
Cited by 1 | Viewed by 2184
Abstract
Traumatic brain injuries (TBIs) are a leading cause of mortality and morbidity, particularly in forensic settings where determining the cause of death and timing of injury is critical. Glial fibrillary acidic protein (GFAP), a biomarker specific to astrocytes, has emerged as a valuable [...] Read more.
Traumatic brain injuries (TBIs) are a leading cause of mortality and morbidity, particularly in forensic settings where determining the cause of death and timing of injury is critical. Glial fibrillary acidic protein (GFAP), a biomarker specific to astrocytes, has emerged as a valuable tool in post-mortem analyses of TBI. A PRISMA-based literature search included studies examining GFAP in human post-mortem samples such as brain tissue, cerebrospinal fluid (CSF), serum, and urine. The results highlight that GFAP levels correlate with the severity of brain injury, survival interval, and pathological processes such as astrocyte damage and blood–brain barrier disruption. Immunohistochemistry, ELISA, and molecular techniques were commonly employed for GFAP analysis, with notable variability in protocols and thresholds among studies. GFAP demonstrated high diagnostic accuracy in distinguishing TBI-related deaths from other causes, particularly when analyzed in CSF and serum. Furthermore, emerging evidence supports its role in complementing other biomarkers, such as S100B and NFL, to improve diagnostic precision. However, the review also identifies significant methodological heterogeneity and gaps in standardization, which limit the generalizability of findings. Future research should focus on establishing standardized protocols, exploring biomarker combinations, and utilizing advanced molecular tools to enhance the forensic application of GFAP. Full article
(This article belongs to the Collection New Advances in Molecular Toxicology)
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11 pages, 246 KiB  
Review
Post Mortem Molecular Biomarkers of Asphyxia: A Literature Review
by Matteo Antonio Sacco and Isabella Aquila
Int. J. Mol. Sci. 2024, 25(21), 11607; https://doi.org/10.3390/ijms252111607 - 29 Oct 2024
Cited by 2 | Viewed by 2228
Abstract
Asphyxia is a critical condition characterized by inadequate oxygen supply to the body. Post mortem diagnostics of asphyxia present significant challenges in forensic pathology, particularly when there are equivocal signs during autopsy or uncertain circumstantial data. The identification of biochemical biomarkers that indicate [...] Read more.
Asphyxia is a critical condition characterized by inadequate oxygen supply to the body. Post mortem diagnostics of asphyxia present significant challenges in forensic pathology, particularly when there are equivocal signs during autopsy or uncertain circumstantial data. The identification of biochemical biomarkers that indicate asphyxia has emerged as a promising area of research, as these markers can provide vital insights into the physiological changes occurring at the cellular level during asphyxiation. We performed a review of the scientific literature on the search engines Pubmed and Scopus in order to assess the state of the art on this topic. The aim of this study is to analyze which are the most promising markers and methods in the post mortem diagnosis of asphyxia. The literature review highlighted the great potential that molecular investigations can have in the analysis of this type of death, especially considering that hypoxia determines strong biochemical alterations in response to cellular stress. These changes are marked by specific biochemical alterations, which can be detected through various advanced technologies and methodologies, including mass spectrometry, immunohistochemistry, and metabolomic profiling. The review evidenced a combination of markers that can be used for diagnostic purposes in various cases, including mechanical asphyxia, carbon monoxide (CO) poisoning, perinatal asphyxia, and drowning analysis. However, we highlight that, to date, there are still no standard protocols for forensic biochemistry in asphyxia. By scrutinizing the reliability of identified biomarkers and their potential to reshape forensic investigative practices, this research aims to elucidate the critical role that post mortem biochemical analysis can play in diagnosing asphyxia, ultimately contributing to a more nuanced understanding of death-related scenarios and the development of standardized protocols in forensic examinations. Full article
(This article belongs to the Special Issue New Perspectives on Biology in Forensic Diagnostics)
21 pages, 883 KiB  
Review
microRNAs as New Biomolecular Markers to Estimate Time since Death: A Systematic Review
by Vincenzo Cianci, Cristina Mondello, Daniela Sapienza, Maria Cristina Guerrera, Alessio Cianci, Annalisa Cracò, Francesco Luppino, Vittorio Gioffrè, Patrizia Gualniera, Alessio Asmundo and Antonino Germanà
Int. J. Mol. Sci. 2024, 25(17), 9207; https://doi.org/10.3390/ijms25179207 - 24 Aug 2024
Cited by 2 | Viewed by 1775
Abstract
Estimating the post-mortem interval is still one of the most complex challenges in forensics. In fact, the main tools currently used are burdened by numerous limitations, which sometimes allow the time of death to be placed only within too large time intervals. In [...] Read more.
Estimating the post-mortem interval is still one of the most complex challenges in forensics. In fact, the main tools currently used are burdened by numerous limitations, which sometimes allow the time of death to be placed only within too large time intervals. In recent years, researchers have tried to identify new tools to try to narrow down the interval within which to place the time of death; among these, the analysis of microRNAs seems to be promising. An evidence-based systematic review of the literature has been conducted to evaluate the state of the art of knowledge, focusing on the potential correlation between miRNA degradation and PMI estimation. The research has been performed using the electronic databases PubMed, Scopus, and WOS. The results allowed us to highlight the usefulness of miRNAs both as markers for PMI estimation and for normalization, especially due to their stability. In fact, some miRNAs remain particularly stable for long periods and in different tissues, while others degrade faster. Furthermore, there are numerous factors capable of influencing the behavior of these molecules, among which the type of tissue, the cause of death, and the circadian rhythm appear to be the most relevant. Despite the promising results of the few articles present in the literature, because of the numerous limitations they are burdened by, further research is still necessary to achieve more solid and shareable results. Full article
(This article belongs to the Special Issue Advances in Molecular Forensic Pathology and Toxicology: An Update)
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15 pages, 1291 KiB  
Review
Sudden Cardiac Death, Post-Mortem Investigation: A Proposing Panel of First Line and Second Line Genetic Tests
by Fabio Del Duca, Alessandro Ghamlouch, Alice Chiara Manetti, Gabriele Napoletano, Elena Sonnini, Biancamaria Treves, Alessandra De Matteis, Raffaele La Russa, Mary N. Sheppard, Vittorio Fineschi and Aniello Maiese
J. Pers. Med. 2024, 14(5), 544; https://doi.org/10.3390/jpm14050544 - 20 May 2024
Cited by 5 | Viewed by 2653
Abstract
Investigating the causes of Sudden cardiac death (SCD) is always difficult; in fact, genetic cardiac conditions associated with SCD could be “silent” even during autopsy investigation. In these cases, it is important to exclude other aetiology and assist to ask for genetic investigations. [...] Read more.
Investigating the causes of Sudden cardiac death (SCD) is always difficult; in fact, genetic cardiac conditions associated with SCD could be “silent” even during autopsy investigation. In these cases, it is important to exclude other aetiology and assist to ask for genetic investigations. Herein, the purpose of this review is to collect the most-implicated genes in SCD and generate a panel with indications for first line and second line investigations. A systematic review of genetic disorders that may cause SCD in the general population was carried out according to the Preferred Reporting Item for Systematic Review (PRISMA) standards. We subsequently listed the genes that may be tested in the case of sudden cardiac death when the autopsy results are negative or with no evidence of acquired cardiac conditions. To make genetic tests more specific and efficient, it is useful and demanded to corroborate autopsy findings with the molecular investigation as evident in the panel proposed. The genes for first line investigations are HCM, MYBPC3, MYH7, TNNT2, TNNI3, while in case of DCM, the most implicated genes are LMNA and TTN, and in second line for these CDM, ACTN2, TPM1, C1QPB could be investigated. In cases of ACM/ARVC, the molecular investigation includes DSP, DSG2, DSC2, RYR2, PKP2. The channelopathies are associated with the following genes: SCN5A, KCNQ1, KCNH2, KCNE1, RYR2. Our work underlines the importance of genetic tests in forensic medicine and clinical pathology; moreover, it could be helpful not only to assist the pathologists to reach a diagnosis, but also to prevent other cases of SCD in the family of the descendant and to standardise the type of analysis performed in similar cases worldwide. Full article
(This article belongs to the Special Issue Clinical Advances in Functional Genomics)
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14 pages, 1296 KiB  
Review
Dating Skin Lesions of Forensic Interest by Immunohistochemistry and Immunofluorescence Techniques: A Scoping Literature Review
by Luca Tomassini, Massimo Lancia, Roberto Scendoni, Anna Maria Manta, Daniela Fruttini, Erika Terribile and Cristiana Gambelunghe
Diagnostics 2024, 14(2), 168; https://doi.org/10.3390/diagnostics14020168 - 11 Jan 2024
Cited by 3 | Viewed by 2951
Abstract
Wound age estimation is a significant issue in forensic pathology. Although various methods have been evaluated, no gold standard system or model has been proposed, and accurate injury time estimation is still challenging. The distinction between vital skin wounds—i.e., ante-mortem lesions—and skin alterations [...] Read more.
Wound age estimation is a significant issue in forensic pathology. Although various methods have been evaluated, no gold standard system or model has been proposed, and accurate injury time estimation is still challenging. The distinction between vital skin wounds—i.e., ante-mortem lesions—and skin alterations that occur after death is a crucial goal in forensic pathology. Once the vitality of the wound has been confirmed, the assessment of the post-trauma interval (PTI) is also fundamental in establishing the causal relationship between the traumatic event and death. The most frequently used techniques in research studies are biochemistry, molecular biology, and immunohistochemistry (IHC). Biochemical methods take advantage of the chemical and physical techniques. A systematic literature search of studies started on 18 February 2023. The search was conducted in the main databases for biomedical literature, i.e., PubMed and Scopus, for papers published between 1973 and 2022, focusing on different techniques of immunohistochemistry and immunofluorescence (IF) for estimating the PTI of skin wounds. The present study involves a comprehensive and structured analysis of the existing literature to provide a detailed and comprehensive overview of the different IHC techniques used to date skin lesions, synthesize the available evidence, critically evaluate the methodologies, and eventually draw meaningful conclusions about the reliability and effectiveness of the different markers that have been discovered and used in wound age estimation. Full article
(This article belongs to the Special Issue Forensic Diagnosis)
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17 pages, 7132 KiB  
Article
Biochemical Toxicological Study of Insulin Overdose in Rats: A Forensic Perspective
by Cunhao Bian, Xin He, Qi Wang, Zhe Zheng, Yongtai Zhang, Hongli Xiong, Yongguo Li, Mingzhu Zhao and Jianbo Li
Toxics 2024, 12(1), 17; https://doi.org/10.3390/toxics12010017 - 23 Dec 2023
Cited by 1 | Viewed by 2950
Abstract
Due to nonspecific pathological changes and the rapid degradation of insulin in postmortem blood samples, the identification of the cause of death during insulin overdose has always been a difficulty in forensic medicine. At present, there is a lack of studies on the [...] Read more.
Due to nonspecific pathological changes and the rapid degradation of insulin in postmortem blood samples, the identification of the cause of death during insulin overdose has always been a difficulty in forensic medicine. At present, there is a lack of studies on the toxicological changes and related mechanisms of an insulin overdose, and the specific molecular markers of insulin overdose are still unclear. In this study, an animal model of insulin overdose was established, and 24 SD rats were randomly divided into a control group, insulin overdose group, and a recovery group (n = 8). We detected the biochemical changes and analyzed the toxicological mechanism of an insulin overdose. The results showed that after insulin overdose, the rats developed irregular convulsions, Eclampsia, Opisthotonos, and other symptoms. The levels of glucose, glycogen, and C-peptide in the body decreased significantly, while the levels of lactate, insulin, and glucagon increased significantly. The decrease in plasma K+ was accompanied by the increase in skeletal muscle K+. The PI3K-AKT signaling pathway was significantly activated in skeletal muscle, and the translocation of GLUT4/Na+-K+-ATPase to sarcolemma was significantly increased. Rare glycogenic hepatopathy occurred in the recovery group after insulin overdose. Our study showed that insulin overdose also plays a role in skeletal muscle cells, mainly through the PI3K-Akt signaling pathway. Therefore, the detection of signaling pathway proteins of the skeletal muscle cell membrane GLUT4 and Na+-K+-ATPase has a certain auxiliary diagnostic value for forensic insulin overdose identification. Glycogen detection in the liver and skeletal muscle is important for the diagnosis of insulin overdose, but it still needs to be differentiated from other causes of death. Skeletal muscle has great potential for insulin detection, and the ratio of insulin to the C-peptide (I:C) can determine whether an exogenous insulin overdose is present. Full article
(This article belongs to the Special Issue Drug Metabolism and Toxicological Mechanisms)
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14 pages, 264 KiB  
Review
An Overview on the Use of miRNAs as Possible Forensic Biomarkers for the Diagnosis of Traumatic Brain Injury
by Giuseppe Davide Albano, Chiara Stassi, Antonina Argo and Stefania Zerbo
Int. J. Mol. Sci. 2023, 24(7), 6503; https://doi.org/10.3390/ijms24076503 - 30 Mar 2023
Cited by 6 | Viewed by 2332
Abstract
Determining the cause of death is one of the main goals of forensic pathology. However, conditions can occur in which common approaches—external inspection, autopsy, histology, etc.—might not be conclusive. With the advancement of molecular biology, several investigative techniques have been developed over the [...] Read more.
Determining the cause of death is one of the main goals of forensic pathology. However, conditions can occur in which common approaches—external inspection, autopsy, histology, etc.—might not be conclusive. With the advancement of molecular biology, several investigative techniques have been developed over the years, and the application as approaches complementary to routine procedures has proved useful in these cases. In this context, microRNA (miRNA) profiling has attracted increasing interest due to these molecules’ ability to regulate physiological and pathological processes. The evidence of differential miRNA expression in both animal models and human samples of traumatic brain injury (TBI) has laid the basis for comprehension of the underlying pathophysiological mechanisms, thus allowing us to identify some of them as possible TBI diagnostic biomarkers. The present narrative review aims to explore the primary miRNAs involved in the mechanisms underlying TBI, which could be considered for future evaluation as possible markers in a post mortem setting. Full article
13 pages, 3781 KiB  
Case Report
Sudden Death without a Clear Cause after Comprehensive Investigation: An Example of Forensic Approach to Atypical/Uncertain Findings
by Simone Grassi, Mònica Coll Vidal, Oscar Campuzano, Vincenzo Arena, Alessandro Alfonsetti, Sabina Strano Rossi, Francesca Scarnicci, Anna Iglesias, Ramon Brugada and Antonio Oliva
Diagnostics 2021, 11(5), 886; https://doi.org/10.3390/diagnostics11050886 - 17 May 2021
Cited by 17 | Viewed by 4294
Abstract
Sudden death (SD) is defined as the unexpected natural death occurred within an hour after the onset of symptoms or from the last moment the subject has been seen in a healthy condition. Brugada syndrome (BrS) is one of the most remarkable cardiac [...] Read more.
Sudden death (SD) is defined as the unexpected natural death occurred within an hour after the onset of symptoms or from the last moment the subject has been seen in a healthy condition. Brugada syndrome (BrS) is one of the most remarkable cardiac causes of SD among young people. We report the case of a 20-year-old man who suddenly died after reportedly having smoked cannabis. Autopsy, toxicology, and genetic testing were performed. Autopsy found a long and thick myocardial bridging (MB) at 2 cm from the beginning of the left anterior descending coronary artery. Furthermore, at the histopathological examination, fibrosis and disarray in myocardial area above the MB, fatty tissue in the right ventricle and fibrosis of the sino-atrial node area were found. Toxicology testing was inconclusive, while genetic testing found a rare missense variant of the TTN gene, classified as likely benign, and a variant of unknown significance in the SLMAP gene (a gene that can be associated with BrS). Hence, despite several atypical features were found, no inference on the cause of the death could be made under current evidence. Full article
(This article belongs to the Section Pathology and Molecular Diagnostics)
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13 pages, 712 KiB  
Review
MicroRNAs as Useful Tools to Estimate Time Since Death. A Systematic Review of Current Literature
by Aniello Maiese, Andrea Scatena, Andrea Costantino, Marco Di Paolo, Raffaele La Russa, Emanuela Turillazzi, Paola Frati and Vittorio Fineschi
Diagnostics 2021, 11(1), 64; https://doi.org/10.3390/diagnostics11010064 - 3 Jan 2021
Cited by 32 | Viewed by 5210
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Progress in the Forensic Diagnosis)
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