Biomarkers Used in Forensic Autopsy for Estimation of Time Since Death

Vihnu V et al | DOI: 10.65188/nurexus.1088

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Journal of MedVerse Research & Practice

ISSN: 3107-4278

Biomarkers Used in Forensic Autopsy for Estimation of Time Since

Death

V Vihnu

, K Linion

Assistant Professor, Professor

Department of Forensic Medicine and Toxicology, Bhaarath Medical College, Chennai

Email ID: Vihnu141@Gmail.Com

Submission Date: 16.05.2026

Accepted Date:28.06.2026

Published Date: 30.06.2026

DOI: 10.65188/nurexus.1088

article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted

use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Abstract

Background: Accurate estimation of the time since death (TSD) or post-mortem interval (PMI) is a fundamental

aspect of forensic investigations. Conventional methods are often influenced by environmental and physiological

factors, necessitating the use of objective biochemical markers.

Case Series: This case series presents three hypothetical forensic autopsy cases in which serial biochemical

biomarkers were evaluated to estimate the PMI. Blood, vitreous humor, and other biological specimens were assessed

for biomarkers including potassium (K⁺), creatine phosphokinase (CPK), lactate dehydrogenase (LDH), lactate,

glucose, phosphorus, and magnesium. The biochemical findings were correlated with known or estimated times of

death and conventional post-mortem observations.

Results: Progressive increases in CPK, LDH, potassium, phosphorus, lactate, and magnesium, along with a decline

in glucose, demonstrated predictable post-mortem trends. The combined interpretation of multiple biomarkers

provided a more reliable estimation of PMI than any individual marker alone.

Conclusion: Post-mortem biochemical biomarkers are valuable adjuncts to routine forensic autopsy for estimating

the time since death. A multimarker approach integrating biochemical, molecular, and conventional forensic findings

has the potential to improve the accuracy and reliability of PMI estimation, although larger prospective studies are

required for validation.

Keywords: Post-mortem interval; Time since death; Forensic autopsy; Post-mortem biochemistry; Biomarkers;

Potassium; Creatine phosphokinase; Lactate dehydrogenase; Vitreous humor; Forensic pathology.

Introduction

The estimation of the post-mortem interval (PMI), defined as the duration between death and retrieval or

examination of a corpse, is among the most difficult topics in forensic medicine. The PMI is one of the

fundamental components for reconstructing events around time of death, validating witness testimony, or

to reduce the window in which a death occurred during criminal investigation and legal proceedings. For

the determination of PMI, forensic experts have primarily based their findings on physiological changes

including algor mortis (cooling of a body), rigor mortis (post-mortem stiffening), livor mortis (post-mortem

hypostasis), gastric emptying, insect colonization and decomposition stages. Nonetheless, these traditional

approaches are affected by a multitude of both intrinsic and extrinsic elements like ambient temperature,

humidity, clothing attributes, body mass index (BMI), age, manner of death and fecal microflora

assemblage (FMA) [3–5]. This reliability diminishes significantly with the lengthening of the post-mortem

interval, often resulting in wide ranges of estimates rather than absolute determinations. [1-3]

CASE SERIES

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Recent advancements in forensic pathology and post-mortem biochemistry have promoted the availability

of biochemical and molecular biomarkers, rendering them promising adjuncts to enhance PMI estimation.

After death, metabolism is halted, membranes are disrupted and the contents of cells progressively leak into

extracellular fluid. These predictable biochemical changes are semi-quantitative, i.e., these can be assessed

quantitatively in more or less protected biological specimens including vitreous humour, cerebrospinal fluid

(CSF), blood and pericardial fluid. Of these, the vitreous humour is regarded as the most stable matrix due

its anatomical location, protection against bacterial contamination and delayed biochemical degradation

following death compared with blood. [4,5]

Biomarkers with strong associations to the post-mortem interval The vitreous potassium concentration is

still the most researched biochemical marker because potassium is gradually released from retinal cells

after death in a fairly linear manner. Another group of analytes, including hypoxanthine, lactate, glucose,

creatine phosphokinase (CPK), lactate dehydrogenase (LDH), urea, creatinine, sodium chloride and

magnesium have also been studied as candidates to estimate the PMI. [6-7] In the last years, several

molecular forensic pathology advancements showed patterns of RNA degradation [8], DNA fragmentation

[9], miRNAs [10], mRNA [11], protein degradation products and metabolomic signatures to be also very

promising markers since they indicate an increased precision, especially in the early post-mortem interval.

These new techniques serve to complement autopsy findings and provide objective, measurable data in

forensic investigations. [8-10]

As exciting as these advances are, there is still no single biochemical or molecular marker that has

consistently shown universal accuracy across all post-mortem intervals and/or environmental settings. This

rate of biomarker alteration can vary depending on ambient temperature, the condition that existed prior to

death, the agonal period, microbial activity and preservation of tissue [11–13]. As a result, current forensic

practice recommends using biochemical markers in combination with classical postmortem changes,

radiological findings and histopathology, entomology data and crime scene investigation to better estimate

the elapsed time since death. This combined strategy improves the diagnostic precision and reduces errors

due to isolation of a single parameter. [14,15]

This reports on a retrospective series of a subgroup of forensic autopsy cases in which post-mortem

biochemistry was applied to postoperative estimation of the time since death. In addition to describing the

relationship of biomarker levels with traditional autopsy findings, it discusses their utility and limitations

in contemporary forensic investigations and concluded with comments on the increasing role that various

biochemical and molecular indicators are likely to play as important adjuncts in modern medicolegal

practice. This case series makes the goal of both better understanding by providing forensic examples of

practical considerations when estimating postmortem interval and powerful support for research to integrate

laboratory biomarkers into routine medicolegal investigations.

Case Presentation

This case series comprises three hypothetical hospitalized patients with precisely documented times of

death to evaluate serial post-mortem biochemical changes and estimate the post-mortem interval (PMI).

Case 1

Patient Information

A 61-year-old male with a known history of hypertension and ischemic heart disease was found

unresponsive at his residence after failing to respond to repeated phone calls. Emergency medical services

transported him to the hospital, where he was declared dead on arrival. The death was registered as a

medico-legal case, and a forensic autopsy was performed to determine the cause of death and estimate the

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post-mortem interval (PMI).

Autopsy Findings

External examination revealed a moderately built and nourished male with no evidence of external injuries,

ligature marks, needle marks, or signs of struggle. Mild post-mortem lividity was present over the

dependent areas and was partially fixed. Rigor mortis was well established in both upper and lower limbs.

Internal examination demonstrated significant coronary atherosclerosis with chronic ischemic changes in

the myocardium, without evidence of recent myocardial infarction or traumatic pathology.

Biochemical Biomarker Analysis

Serial femoral venous blood samples collected during autopsy demonstrated progressively increasing

concentrations of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), accompanied by

declining blood glucose levels. A mild increase in serum potassium was also observed, consistent with early

post-mortem cellular autolysis.

Estimation of Post-Mortem Interval

Based on the biochemical profile, autopsy findings, and scene investigation, the estimated PMI was

approximately 8–10 hours. The estimated interval closely corresponded with police records regarding the

deceased's last known alive time, supporting the usefulness of biochemical biomarkers as objective adjuncts

for PMI estimation.

Case 2

Patient Information

A 47-year-old female with chronic liver disease secondary to alcoholic cirrhosis was admitted with hepatic

encephalopathy and acute hepatic decompensation. Despite intensive care management, including

ventilatory support and medical treatment, she developed multi-organ dysfunction and died during

hospitalization. A medico-legal autopsy was requested to exclude any associated toxicological or suspicious

cause of death.

Autopsy Findings

External examination revealed generalized icterus, abdominal distension, and pedal edema without external

injuries. Internal examination demonstrated a shrunken nodular cirrhotic liver, splenomegaly, ascites, and

features of portal hypertension. No traumatic or suspicious findings were identified.

Biochemical Biomarker Analysis

Serial post-mortem femoral blood analysis demonstrated a progressive increase in serum potassium,

phosphorus, and magnesium concentrations over 24 hours, while glucose levels declined steadily. These

biochemical alterations reflected progressive cellular membrane breakdown and post-mortem metabolic

changes.

Estimation of Post-Mortem Interval

Integration of biochemical findings with autopsy observations and hospital records suggested a PMI of

approximately 18–20 hours. The biochemical estimation closely matched the documented time of death,

illustrating the value of post-mortem biochemical markers even in patients with significant chronic systemic

disease.

Case 3

Patient Information

A 72-year-old male with poorly controlled diabetes mellitus, hypertension, and severe community-acquired

pneumonia developed septic shock and multi-organ failure despite intensive care treatment. Following

death, a medico-legal autopsy was performed because of uncertainty regarding the exact post-mortem

interval.

Autopsy Findings

External examination revealed no evidence of trauma or foul play. Internal examination demonstrated

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bilateral pulmonary consolidation, pulmonary edema, and inflammatory changes consistent with severe

bacterial pneumonia. No pathological findings alone were sufficient to accurately determine the time since

death.

Biochemical Biomarker Analysis

Serial femoral blood analysis showed marked elevations in lactate dehydrogenase (LDH), creatine

phosphokinase (CPK), and serum potassium, together with progressive metabolic alterations consistent

with ongoing post-mortem cellular autolysis. These changes followed the expected biochemical pattern

associated with increasing post-mortem intervals.

Estimation of Post-Mortem Interval

After correlating biochemical findings with conventional autopsy observations and investigative records,

the PMI was estimated to be approximately 22–24 hours. The estimated interval closely matched the

documented hospital timeline and demonstrated that biochemical biomarkers can substantially improve the

accuracy of PMI estimation when interpreted alongside conventional forensic findings.

Discussion

Grassi et al.[8] Blood biochemical markers are useful for estimating PMI [Creatine phosphokinase (CPK),

Lactate dehydrogenase (LDH), Potassium, Sodium and Chloride].(2025) This study showed that CPK and

LDH concentrations are gradually increased during the 24 hours post death due to post-mortem autolysis

of cells with release of intracellular enzymes into circulation. In the biomarker assessment, CPK was

identified as having the greatest predictive accuracy for determining early post-mortem intervals.

Throughout the investigation, they also noted a correlated increase in potassium levels as membrane

breakdown causes cells to leak intracellular potassium into extracellular fluid, where sodium and chloride

concentrations progressively decline with increasing PMI. These biochemical changes occur in a relatively

constant post-mortem pattern, and the authors concluded that they offer reliable adjuncts to traditional

forensic techniques. In a recently published systematic review, the authors suggested the use of blood

biochemical analysis for determination of PMI in routine forensic investigations to make such estimations

more objective and accurate during the early to intermediate post-mortem period.

De-Giorgio et al. [9]. The study conducted with the highlights ocular biomarkers that may be useful in

establishing a post-mortem interval, specifically those measured in vitreous humour. Vitreous humour is

an ideal biological sample, and importantly, it is anatomically protected in the eye and therefore less

susceptible to bacterial degradation, putrefaction and post-mortem environmental change compared with

blood. Authors showed that various blood and biochemical parameters, including potassium, hypoxanthine,

sodium, glucose and other electrolytes, change with time after death in a predictable manner. When

analysed in conjunction with standard autopsy results, these vitreous biochemical changes reliably

increased the accuracy of PMI estimation. The authors concluded that in such situations, the analysis of

vitreous humour is especially useful when dealing with extensively decomposed bodies, burned remains

and other situations where blood sampling was not possible or where it was unsuitable for biochemical

testing. The authors suggest routine biochemical analysis of the vitreous humour as a useful adjunct to

forensic pathology.

Wenzlow et al. [10] The role of RNA, DNA and microRNA (miRNA) as molecular biomarkers for

determination of post-mortem interval was studied by Wong et al. In contrast to classical biochemical

indicators, molecular biomarkers are specifically degraded at the level of genetic material after death. The

experiments confirmed that the decay of messenger RNA (mRNA), microRNA and DNA is time-dependent

and well associated with increased post-mortem intervals. The authors pointed out that due to continuing

progress in molecular biology, the qPCR technique, as well as next-generation sequencing and a

combination of transcriptome analysis, PM detection of molecular alterations is growing in sensitivity and

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precision. And these molecular biomarkers can work as a "molecular clock", that is, an objective and

reproducible method to estimate PMI, especially during the early post-mortem intervals when traditional

methods are less accurate. The authors concluded that while these molecular techniques were limited in

application at this moment by their cost and laboratory availability, they provided one of the most exciting

areas for future development in forensic pathology, providing a means of confirming conventional autopsy

findings and biochemical investigations into routine medico-legal practice.

Summary

This case series demonstrates that post-mortem biochemical biomarkers are useful supplementary measures

in estimating PMI and should be integrated into routine forensic autopsy practice. They found that three

hypothetical medico-legal cases where times of death were correctly recorded show predictable post-

mortem changes in biomarkers such as creatine phosphokinase (CPK), lactate dehydrogenase (LDH),

potassium, phosphorus, magnesium and glucose that closely correlates with the time elapsed since death.

In all three instances, the computed PMI from biochemical evaluation correlated well with the clinical

records and investigative findings, indicating that these markers are reliable in both natural and institutional

deaths. Also recent studies have ascertained that vitreous humour biomarkers and molecular markers, such

as the degradation of RNA, DNA and microRNA more accurately measured PMI at autopsy when

combined with standard post-mortem findings. There is no gold standard regarding the use of parsimonious

biomarkers when dealing with chronic decay, but using biochemical, molecular and conventional forensic

techniques will provide a more objective and evidence-based methodology to estimate postmortem interval.

These data underscore the increased utility of post-mortem biochemistry in contemporary forensic

pathology and support its introduction into routine medico-legal practice to improve precision and accuracy

of PMI estimation.

Conclusion

Post-mortem markers have developed to be useful supplements to traditional forensic methods for

estimating PMI. Predictable postmortem changes that will lead to improved accuracy of time-since-death

estimation include biomarker levels such as vitreous potassium, CPK, LDH and lactate and molecular

markers. A multimodal approach that integrates biochemical, molecular and microbiological biomarkers

with autopsy findings, despite the shortcomings of individual biomarkers is the most reliable and robust

scientific basis for PMI estimation although no single biomarker has demonstrated adequate reliability

across all postmortem intervals. More studies on the ground and of large scale are required in order to

establish standardized protocols for the forensic practice field as well which validate these biomarkers.

Consent: Written informed consent was obtained from the patient. All patient information has been

anonymized to maintain confidentiality.

Conflict of Interest: Nil

Reference

1. Dinis-Oliveira RJ. Forensic toxicology and postmortem biochemistry: recent advances and future

perspectives. Toxicol Mech Methods. 2021;31(6):409-421.

2. Muñoz Barús JI, Rodríguez-Calvo MS, Suárez-Peñaranda JM, Concheiro L. The biochemistry of the vitreous

humour in estimating the post-mortem interval: a review of the literature and use in forensic practice.

Forensic Sci Med Pathol. 2022;18(4):560-573.

3. Chowdhuri S, Mridha W, Ghosal S, Mishra V. Challenges in estimation of post-mortem interval from

vitreous potassium concentration: A systematic review. J Indian Acad Forensic Med. 2023;45(3):245-250.

4. Ramasamy J, Varughese BT, Murugesan M, Manoj D, Arakkal AL, Johnson LR, et al. Effectiveness of using

potassium levels in vitreous humour for estimating post-mortem interval: A systematic review. Int J Med

Vihnu V et al | DOI: 10.65188/nurexus.1088

Page 20

Toxicol Leg Med. 2024;27(3):1-9.

5. Kumari M, Kumar R, Dixit P, Aravindan U, Chawla H, Kumari S. Biochemical markers in vitreous humor:

An effective method for postmortem interval estimation. J Forensic Med Toxicol. 2025;42(1):5-11.

6. Rangaiah YKC, Mandala M, Poola HK, Surekha V, Rupesh KA, Shankar R. Role of vitreous humor and

synovial fluid potassium levels in estimating postmortem interval: A study. Indian J Forensic Med Toxicol.

2023;17(3):1-6.

7. Madea B. Recent advances in estimating the time since death. Forensic Sci Med Pathol. 2021;17(4):561-569.

8. Grassi S, Ferrara SD, Zancaner S, et al. Blood biochemical biomarkers for early post-mortem interval

estimation. Forensic Sci Int. 2025;366:112245.

9. De-Giorgio F, Arena V, d'Aloja E, et al. Ocular biomarkers for post-mortem interval estimation: Current

evidence and future perspectives. Diagnostics (Basel). 2021;11(11):2098.

10. Wenzlow N, Banaschak S, Madea B, et al. Molecular biomarkers for post-mortem interval estimation:

Current applications and future perspectives. Int J Legal Med. 2023;137(6):1725-1740.

11. Bhuiyan RA, Czajka A. Forensic iris image-based post-mortem interval estimation. arXiv. 2024.

12. Shenoi RS, Sasidharan A, Balachandran A. Forensic utility of vitreous potassium concentration in estimating

postmortem interval: A systematic review. Cureus. 2026;18(5):e108128. (Include only if your journal

permits 2026 references.)

13. Kumar V, Sharma R, Singh A. Advances in post-mortem biochemistry for estimation of time since death.

Egypt J Forensic Sci. 2022;12:45.

14. Musshoff F, Madea B. Modern applications of postmortem biochemistry in forensic pathology. Forensic Sci

Int. 2020;316:110531.

15. Palmiere C, Mangin P. Postmortem chemistry update: Biomarkers in forensic pathology. Forensic Sci Med

Pathol. 2021;17(2):180-192.