Recent Advances in Forensic Medicine at the Horizon of Omics Sciences and New Biomarkers for Postmortem Interval Estimation

Abstract

Objectives

To present recent advances in forensic sciences with omics sciences and new biomarkers for postmortem interval (PMI) estimation.

Materials and methods

We conducted a narrative review screening PubMed and Scopus databases in the last 10 years (2015-2025) with the following keywords in the title and abstract: "postmortem interval" OR "post-mortem interval" AND "proteomics" OR "proteomic" OR "metabolomics" OR "metabolomic" OR "transcriptomic" OR transcriptomics" OR microRNA" OR "microRNAs" OR "lipidomic".

Results

Conventional methods of postmortem interval estimation are presented. Some of the most important studies and molecular techniques in genomics, transcriptomics, proteomics, metabolomics, lipidomics, old and new biomarkers for postmortem interval estimation are summarized. Single-omics or multi-omics, critical issues like data reproducibility and interpretation, judicial validity according to Daubert standard and ethical issues of PMI research are discussed.

Conclusions

Postmortem interval estimation continues to be one of the most disputed issues of forensic medicine. Conventional methods for PMI estimation still offer a solid bench for practical means. As single-omics and multi-omics research continues to progress, we will likely discover new biomarkers and innovative techniques. Efforts will focus on identifying biomarkers that can deliver reliable and predictable outcomes, thereby facilitating their general acceptance and admissibility in legal proceedings.

INTRODUCTION

Death date or time of death is probably the most frequently investigated issue in forensic medicine throughout time, which is due not only to its juridical importance but also to the early comprehension that there is no unique reliable method that allows an accurate estimation, no matter what environmental conditions or causes of death are present. Several terms are used; thus, "time since death" has a better connection to the period from discovery of a body back to death (an abductive logical approach) or "postmortem interval" moving from death to discovery (an inductive approach); we will use postmortem interval (PMI) in our study. Police most often start an investigation from the date of death, asking "When?" while trying to configure a reliable circle of suspects at a fast pace. This question generates a conventional method approach from the coroner or the forensic pathologist to allow an estimate of PMI at the scene, traditionally separating immediate, early and late PMI (1, 2).

Police always pay close attention to all items that allow a PMI estimation when called on the scene: clothes, labels, coins, personal belongings, different items, paper writings, photos (3), captured images or video footage, reliable witness statement or the last known phone call (4) or the last time that the person has been seen (5).

If a refined answer is to be given, the short question "When?" often hinders a complex answer, which in many cases finds solutions in laboratories where translational omics sciences ground applicable results in a teamwork effort: forensic pathologists, forensic scientists, biologists, physicists, biochemists, etc.

Accurate recording of environmental conditions is crucial for PMI estimation, as these factors can cause significant changes in how a body decomposes across different regions, even when circumstances appear similar (4). Outdoor factors that intervene include body size (6), temperature (7), burial conditions – i.e., body exposure (8), clothes, burial conditions (9) (depth at which the cadaver was buried, soil and vegetational changes), bacterial activity (9, 10), decomposition stages (11-14), water submersion, insect activity (15), weather conditions, humidity, oxygen availability, inflicted trauma and cause of death, taphonomy changes (16), (17) chemical substances, etc. Similarly, indoor conditions like temperature and dryness may also modify decomposition rate. Eventually, forensic science provides "a "time bracket" of probability" of PMI (1) in its effort to assess the time of death.

METHODS

This review outlines current PMI estimation methods and emerging omics biomarkers for improved accuracy. We have screened omics sciences methods and results in the last 10 years, between 2015-2025, since protein degradation methods had a novel approach in 2015 (18, 19), and most protein degradation studies were published in this time period in as much as studies on human samples started to be conducted and presented in this period as original articles.

For this review we searched PubMed and Scopus databases to find relevant studies or scientific positions to be cited using the following keywords in the title and abstract: "postmortem interval" OR "post-mortem interval" AND "proteomics" OR "proteomic" OR "metabolomics" OR "metabolomic" OR "transcriptomic" OR transcriptomics" OR microRNA" OR "microRNAs" OR "lipidomic".

We made correlations between conventional methods and omics sciences methods. Justice uses biological results of validated methods subjected to the Daubert criteria. Admissibility in a court of law is a judicial task (20). Figure 1 presents a Graphic abstract depicting methods and correlations used for this review.

FIGURE 1.

Graphic abstract depicting methodology, methods and correlations used to refine the postmortem interval (PMI) as presented in this narrative review. The omics sciences tree is depicted. Laboratory as personnel, equipment and methods to manage large data are figured. Post-translational modifications may alter protein stability and impact PMI estimation (original creation using Mind the Graph).

RESULTS

1. Conventional methods to estimate the postmortem interval

Conventional approaches for estimating PMI (2, 21) rely on visual examination, observation of physical alterations like postmortem changes, temperature assessments and analysis of local arthropod development stages and succession. These techniques are used at the scene to estimate both the highest and lowest possible PMI values (22). Usually, conventional methods are conclusive in a court of law (20).

Temperature is most probably the current gold standard in forensic practice to estimate PMI based on a model (23, 24). Some authors space themselves from the model and for practical reasons add subjective correction factors in nonstandard conditions (25).

Conventional methods separate three decomposition phases (21). The immediate postmortem phase occurs in the initial 2–3 hours between somatic death and cellular death. This period, marked by changes to the eyes (26) and skin (27), is distinguished by the beginning of autolysis and the presence of supravital reactions (3).

Early postmortem phase (fresh stage), 3-72 hours, with the process of autolysis that continues (1, 20). Rigor mortis begins 2-6 (8) hours till 24 hours (28), livor mortis begins two hours and becomes fixed over 12 hours (29), and algor mortis which states as a rule of thumb a decrease of 1.5 degrees F every hour (30) – all offer a practical PMI estimate.

In the late postmortem phase (>73 hours), the accuracy and precision of the PMI estimate decrease (2, 3). The body decomposes passing through five decomposition stages (31-36): fresh (installation 24 hours-5 days, oviposition, no odor), early decomposition (installation 1-5 days-1-2 months, skin slippage, maggots, greenish bloating abdomen, brownish extremities), advanced decomposition (installation 2-9 months abdominal collapse, loose desiccated leathery skin, pupal activity, adipocere), skeletonization (installation 2-9 months till years, dry greasy bones) and extreme decomposition (installation one year-1.5 years, bones with erosion exposed to environment, metaphyseal loss at 5.5 years).

Other authors consider the following five stages: an initial decay 1-3 days, putrefaction 4-10 days, black putrefaction 10-20 days, butyric fermentation 20-50 days and dry decay 50-365 days (37).

In this late postmortem phase, development stages and entomology mark an important step in narrowing the late postmortem interval (2, 21, 38).

2. Biomarkers, methods and techniques of the molecular approach to PMI

Following the use of observational techniques and conventional methods for estimating PMI, researchers begin searching for biological biomarkers. This search aims not only to interpret previous observations but also to advance fundamental scientific research at the tissue and cellular levels. A biological marker (biomarker) is "A defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention" (39).

Researchers have long sought biomarkers, as the smell of decomposition releases more than 400 volatile organic compounds. These compounds attract insects but also present a challenge for biochemists and forensic scientists searching for useful biomarkers to estimate the postmortem interval (40). Additional cadaveric odor biomarkers have been identified in this area, such as cadaverine and putrescine found in brain tissue (41), along with various other compounds produced during decomposition (42-44). New biomarkers emerged by studying both animal models and deceased human bodies. Discovering new biomarkers is critical to the development of medical sciences and to the postmortem interval estimate.

Searching the literature listed for old and new biomarkers, we observe that tissues like skeletal muscle, myocardium, and brain are commonly used for early PMI estimation, to a lesser extent, gingival tissue, liver, pancreas, lung, thyroid, kidney tissue, blood, vitreous humor and decomposition fluids; on the other hand, bones are used for long PMI estimation. Table 1 presents a selection of old and new biomarkers for PMI estimation.

As we may easily appreciate, most studies focused on early PMI (interval 0–10 days from death), and this could be explained by two factors: (1) most corpses that are found and enter the investigation are fresh and (2) the police need to assess as quickly as possible a PMI estimate.

TABLE 1.

Some old and new biomarkers for PMI estimation, their original tissues and the methods used for quantification

3. Omics sciences

Conventional methods of estimating PMI coexist with laboratory methods and techniques developed throughout time, and try to refine PMI estimation, especially when environmental factors have altered postmortem interval dynamics.

Animal models, the possibility to have a direct access through forensic autopsy to cadaveric samples, development of extraction techniques, i.e., protein extraction, metabolites and any other biological material from animal or human tissues source, development of the laboratory setup, equipment technology, methods (Table 2) and high skill laboratory personnel, altogether coordinate to emerge a new horizon of collective sciences, named omics sciences connected in a multidisciplinary field that brings together characterization and quantification of biological molecules such as genes (genomics), RNA (transcriptomics), proteins (proteomics), metabolites (metabolomics), lipids (lipidomics). Omics sciences generate a more comprehensive understanding of molecule types and regulatory mechanisms of cellular machinery (56). Omics sciences are closely linked to advanced technologies like high-throughput sequencing, mass spectrometry, Fourier Transform Infrared (FTIR) spectroscopy. By combining the expertise of specialists, these approaches aim to identify and analyze a wide range of molecules found inside our cells, tissues and body structures. Table 2 presents the methods and techniques used for the molecular approach to PMI interval estimation.

TABLE 2.

Methods and techniques used for the molecular approach to PMI interval estimation.

Genomics

Genomics techniques investigate the genome at the somatic and germline levels. Microarray technology (57), first-generation sequencing, second-generation sequencing, also next-generation sequencing (NGS) (58) and third-generation sequencing (TGS) (59) can sequence the entire genome in each sample.

When an organism dies, cellular nucleases degrade chromosomal DNA into smaller fragments. As the postmortem interval increases, chromatin breaks down, and high molecular weight DNA gradually vanishes. DNA degradation is of limited value to forensic investigations requiring an estimation of PMI (60).

Transcriptomics

Transcriptomes refer to mRNA transcripts, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNA) and quantify RNA molecules transcribed from DNA (61). RNA microarrays can profile different expressed genes and identify markers.

Temperature is crucial when studying RNA degradation: environmental temperature of the body (and samples) and laboratory temperature. mRNA appears more unstable than DNA and other proteins in the estimation of PMI (62).

It has been observed that the biological clock halts at death, leading to a noted correlation between the expression of circadian rhythm genes (NR1D1 and BMAL1) and the estimation of postmortem intervals. Ratios NR1D1/BMAL1 and BMAL1/NR1D1 may potentially be used to estimate postmortem interval and the time of death itself (63).

Circadian clock gene expressions can change due to internal factors like brain injury or aging, which should be documented.

Proteomics

Proteomics (Mark Wilkins, 1995) initiates the study of the proteome (more than 100.000 proteins in a cell). The first use of degradation of proteins in human samples as a novel method for postmortem interval estimation was in 2016-2017 using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western Blotting (18, 19). Mass-spectrometry, including MS/MS, high resolution Orbitrap, matrix-assisted laser desorption/ionization time of flight, MALDI-TOF-TOF, FTIR spectroscopy, liquid chromatography mass spectroscopy, LC-MS/MS, gas-chromatography mass-spectrometry GC-MS/MS are very advanced technologies able to separate proteins. We identified six studies using human tissues in the last 10 years, 2015-2025. Figure 2 presents an infographic depicting human studies on PMI (with references), published between 2016-2022 in the mentioned study period (see methods).

Desmin and polyubiquitin prove in vitreous humor to have a degrading predictability between 15–160 hours PMI and 42–160 hours PMI, respectively (70).

Collagen protein type I has a high discriminatory ability to differentiate PMI up to 20 years, while non-collagenous proteins are better biomarkers in long PMI because of better longevity and stability than collagenous proteins, e.g., collagen protein type I (71).

FIGURE 2.

Infographic that presents human studies on PMI estimation research (2016-2022): authors with references (66,67,68,69), sample type (skeletal muscle for short PMI and bone for long PMI), and methods used (original creation using Mind the Graphic).

Metabolomics

Metabolites (MW <1.500 Da) are molecules that participate in cell metabolism as energy sources, signaling and modulatory molecules, and are not genome, transcriptome, proteome, or metals (56). Methods used to identify, quantify and characterize biomolecules are spectroscopy, FTIR, Raman and NMR spectroscopy, all non-destructive, GC-MS, LC-MS and MS/MS.

Metabolic profiles of the tissues consistently vary with the manner and probably with the cause of death and correlated PMI (72).

Most PMI studies use animals and most accurately identify skeletal muscle metabolites when PMI is under 24 hours (73, 74).

As for intermediate and late PMI, N-acetylneuraminate is consistent for PMI < 19 days (75) as well as 1-methylnicotinamide, choline phosphate, uracil, tyrosine, threonine, lysine, 1H NMR with superior results for PMI estimation (76). Human eye (aqueous and vitreous humor) are useful samples for metabolites research in PMI estimation with FTIR separation (77). There is a need for standardization of metabolomic methods in PMI evaluation (78).

Lipidomics

Lipidome is the complete profile within a cell and is a part of the metabolome within that cell with specific and unique functions. Research methods are mass spectrometry MS, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. Lipids connected to the hydroxyapatite of bone matrix last longer and are useful for postmortem interval estimation (79).

Lipidomic methods are rarely used for short postmortem intervals because lipids stay stable after death. Phosphatidylcholines found in trabecular bone show a steady decline as the postmortem interval increases, but they can persist at low concentrations for decades (80).

Sterols (cholesterol, 5α-cholestanol and cholestanone) SFA (C18:0 and C16:0) and UFA (C18:1 and C18:2) are found by GC–MS/MS in muscle tissue (81), lysophosphatidylcoline (PI) and phosphatidylcoline (PC) in bone using LC–MS (82), SFA, UFA and bile acids in textiles in contact with decomposing remains using GC–MS/MS (83), SFA (C14:0, C16:0 and C18:0), hydroxy fatty acids and fatty acid salts using ATR-FTIR and chemometrics proving that ATR-FTIR and chemometrics may explore the estimation of PMI in adipose tissue and the effect of temperature (84).

DISCUSSIONS

1. Single omics or multi-omics?

Researchers consistently strive to identify novel biomarkers that provide better correlations, improved predictability, and lower costs. Laboratory endowment and acquisitions, especially of high technologies (i.e., MALDI-TOF, LC-MS/MS, Mass Spectrometry, etc) call for new biomarkers in the first instance because such technologies have improved analytics, high discrimination, good accuracy, etc. but also because these advanced technologies generate support for research, data communication and scientific connections (teamwork) in complementarity with scientists, laboratories and other sciences (e.g., forensic medicine/biology, forensic medicine/physics, etc) or inter-disciplinarity (e.g., forensic medicine/justice, etc).

Biological evidence becomes proof in a court of law only when accepted and validated by the court. For instance, if PMI is scientifically determined using various approaches, including conventional techniques and omics sciences (e.g., proteomics and metabolomics), the assumption of admissibility in a court of law increases.

Single-omics studies still dominate literature. Single omics proteomics biomarkers (e.g., vinculin, desmin, cardiac troponin T (cTnT), actin, myosin, titin, nebulin, calpain-1, calpain-2, SERCA-1) prove to be valid and valuable in time (18,19). Nevertheless, researchers continue to identify additional biomarkers. Using potent technologies, more biomarkers are identified in a single study: e.g., mass-spectrometry proteomics 275 human proteins (66), iTRAQ-based proteomics 159 pig proteins (85), metabolome in animal models 145 mice metabolites (86) or 51 pig metabolites (87).

At least till now, single-omics metabolites (metabolomics, lipidomics) prove to be more stable for short PMI than proteins (proteomics), which look more useful for longer PMI.

Multi-omics generates extensive data, which can be advantageous when combined with machine learning (89,90) or mathematical modeling (91) v. single omics.

Multi-omics, combining with bioinformatics and artificial intelligence, may offer a better understanding of the cause of death (92) and possibly a better estimate for long PMI, and overcome certain challenges posed by environmental or postmortem conditions.

Nevertheless, multi-omics presents challenges such as very large data management, heterogeneity (93); computational methods, deep generative models, and machine learning development to offer solutions (94).

The use of the "Forensomics" concept confirms the importance of a multi-omics approach in PMI estimation (95).

When discussing single-omics v. multi-omics, it is also important to consider the research capabilities for multi-omics and personnel specialization.

2. Critical issues

Data reproducibility and interpretation

We found several key factors that may have implications on biomarkers and PMI estimates: species, gender, age group, environmental conditions (weather, temperature, etc) (96), death circumstances, cause of death, taphonomy modifications, clothing, if any, methods of exhumation, laboratory transportation conditions (refrigerate sample/non-refrigerate sample), etc. We also noticed that aspects related to laboratory flow, such as sample types, sample heterogeneity, sample size variations, repeated analyses to frame PMI either in the laboratory from the samples or from the cadaver refrigerated for the study over a longer period, must be carefully observed, documented and controlled. Ethical considerations in research are essential.

A solution for data reproducibility may be a larger cohort of subjects to evaluate selected metabolites or biomarkers and quantification using mathematical models for PMI estimation (91).

As we observed there is not yet a correlation between the decomposing phases of the cadaver we may determine when applying conventional methods and PMI intervals as studied in laboratory by omics sciences: for instance short postmortem interval as defined in laboratory studies (no longer than seven days) is included partially in the early postmortem decay phase and partially in the late postmortem decay phase whereas intermediate postmortem interval (usually defined in laboratory studies until 120 days) and late postmortem interval (more than 120 days) belonging both to the late postmortem phase of body decay.

Laboratory-defined PMI intervals do not align body decay phases. A solution would be a correlated conceptualization between biological phases of body decomposition/decay and postmortem intervals (short, intermediate, long PMI), as far as I see, as an initiative that may spring from the laboratory researchers. However, biomarker results do not have implications on data validation or accuracy, but aligning body decay phases and postmortem intervals into a unified perspective will help a better understanding of the decay process and will allow complementary forensic pathologists/coroners and biologists/biochemists/physicists, etc. A multidisciplinary approach to solve the gap between decomposition theory and forensic research on postmortem intervals will be very useful (97). A planned approach to sample collection as the autopsy progresses is also essential (98).

Judicial validity of postmortem interval estimation in a court of law

All biological traces within the body or outside of a body may become evidence in a court of law (99).

Data acquired through different omics techniques and the admissibility of scientific evidence in a court of law are tested by using Frye or Daubert standards.

The "Daubert standard" provides a framework to use judicially, which requires assessment of the evidence to ensure scientific validity (as prior tested), reproducibility (as publication and peer review), predictability (as potential error rate acknowledged), reliability (as clear standards), and admissibility (as acceptance) in a court of law (100, 101).

The Daubert Standard supplanted the "Frye Standard", which focused primarily on the general acceptance of scientific evidence within related scientific fields (102, 103).

We represented in Figure 1 the admissibility in a court of law of the PMI (estimate by conventional methods, single omics, e.g., proteomics or multi-omics). The obligation to make the burden of proof fall to the expert, forensic pathologist/coroner.

Henssge nomogram (23, 24) and entomological methods meet Daubert criteria (100). Expert competence, knowledge, experience, and skills are essential, as forensic methods, even those that meet Daubert criteria, are effective only when applied correctly by qualified professionals (104).

Predictability, peer review, scientific publications, and scientific community validation (general acceptance), are all factors that contribute to the judicial admissibility of a single omics result based on Daubert standards. Scientific peer-review (test, publication, reproducibility) offers contradictory and general acceptance. However, justice reserves the right to refuse admissibility.

So many biomarkers and methods may generate confusion in a court of law. Many assessed biomarkers or PMIs lack universal acceptance.

Proteomic techniques, including Western blotting and mass spectrometry, fulfill several Daubert criteria, such as scientific validity, reproducibility, and, for certain biomarkers (18, 19), predictability in normal environmental conditions. Predictability is still large due to, e.g., environmental conditions, other conditions as presented already (sampling, transportation, temperature, laboratory flow, etc.), and therefore difficult to control. There is a need for standardization from researcher to researcher (reliability). General acceptance in the scientific community depends on completing all standards, but is critical for predictability and standardization.

Laboratory validation methods, good clinical practice of laboratory, trained and specialized personnel, management control protocols (e.g., temperature), service plan for equipment, acquisition of valid markers with international acceptance, scientific publication, etc., are all integrated validation requirements that must be documented if required specifically in a court of law. We must keep in mind that a specific biomarker or method may meet general acceptance, but still not meet admissibility in a particular court of law or in a particular country, because justice itself decides admissibility in its court.

Postmortem interval estimation in a court of law or a professional written report is usually based on a nomogram Henssge, entomology if the case and conventional methods. If legal authorities need a more accurate estimate of the postmortem interval (PMI), laboratory tests are usually performed. Such procedures typically utilize proteomics methodologies that are more commonly accessible within forensic or legal medicine departments. Complementarity may generate the possibility to access other omics sciences like metabolomics (e.g., vitreous humor as a sample from the autopsy analyzed in a physics department using FTIR) and to create a work team to provide reliable information and results. However, protocols between the institutions involved are necessary (e.g., legal medicine/university, department of physics) and official requests must be documented for transparency and if required, in court.

We look forward to following this topic as it continues to be discussed in the coming years.

Ethical issues of PMI research

Ethical aspects of forensic procedures and omics development are always in the researcher's attention. If the forensic pathologist/coroner is the researcher themselves, then ethical aspects are important. Law enforcement forces usually require an autopsy as judicial proof to provide solid evidence in a court of law and informed consent for the autopsy is usually not required. Legal provisions respond to this requirement. However, if research involves procedures, sampling methods, or techniques that are not part of the standard autopsy protocol (whether established by national or international legal provisions), additional considerations must be addressed (e.g., Recommendation EU 1159 (1991) Harmonization of autopsy rules).

Ethical issues concern also ethics connected to forensic or clinical autopsy (105,106), ethics of research on human subjects (full adherence to the Declaration of Helsinki (DoH), 1964-2024 and any national legal provisions) and ethics of research on animal (the 3Rs principles as Replacement, Reduction, Refinement and full adherence to The ethics of research involving animals a guide to the Report, Nuffield Council on Bioethics)

Ethical implications of regulations to follow and procedures for obtaining biological samples for further diagnostics or research, ethical concerns are regarding data privacy for genetic information, storing and further using of sample collections, cultural/religious considerations, minimal invasive procedures, relations between families and healthcare personnel (physicians and technicians) involved in conducting an autopsy. Researchers and forensic experts must consider these ethical issues in all PMI studies, regardless of the method used. It is necessary to present a research protocol and get approval from an Institutional Review Board (Ethics Committee) for any research on PMI estimation. Informed consent should also be secured from the next of kin if human study research implies methods and techniques or sampling that are not specifically required by justice.

Scientific publication with animal models or direct human studies supports peer-review, contradictory or general acceptance implying a deeper knowledge of human biology, medicine progress, increased validity in a court of law, and trans-disciplinarity with translational implications (society-oriented values, family, anthropological and cultural insights), science giving back added value to society.

CONCLUSIONS

Postmortem interval estimation continues to be one of the most disputed issues of forensic medicine and criminalistics. Conventional methods for PMI estimation still offer a solid bench for practical means. Body temperature (nomogram Henssge) is a gold standard for PMI estimation. Entomology offers reliable data in a court of law.

As single-omics and multi-omics research continues to progress, we will likely discover new biomarkers and innovative techniques. Efforts will focus on identifying biomarkers that can deliver reliable and predictable outcomes, thereby facilitating their general acceptance and admissibility in legal proceedings. Ethical considerations are essential in all PMI estimation research involving animal models or human subjects.

Conflicts of interest

none declared.

Financial support

none declared.