Biomarkers Used in Forensic Autopsy for Estimation of Time Since

Thivya R et al | DOI: 10.65188/nurexus.1081

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Journal of MedVerse Research & Practice

ISSN: 3107-4278

Biomarkers Used in Forensic Autopsy for Estimation of Time Since

Death: A Case Series

R Thivya

1

, N Suba

2

,

Assistant Professor, Professor

Department of Ophthalmology, All India Institute of Medical Sciences, Bathinda.

Email ID: thivyaraj@gmail.com

Submission Date: 10.04.2026

Accepted Date:20.05.2026

Published Date: 31.05.2026

DOI: 10.65188/nurexus.1081

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 determination of the postmortem interval (PMI) is one of the most important and

demanding aspects in forensic pathology and medicolegal investigation. Listings like algor mortis, rigor

mortis and livor mortis are often affected by environmental and biological factors and not accurate.

Biochemical and molecular biomarkers have emerged as objective tools over the last decade, driven by

advances in forensic science, to optimize PMI estimation.

Case presentation: This case series reports 3 forensic autopsy cases with early, intermediate and late

postmortem intervals. Comprehensive medicolegal autopsies, which included vitreous humour biochemical

analysis, histopathology, toxicology and molecular biomarker evaluation where applicable. Potassium and

hypoxanthine concentrations (microM) were progressively increased with the advancing postmortem

intervals. In the case of decomposition, molecular biomarkers in terms of RNA degradation profiles and

immunohistochemical markers were also a supplementary means to estimate PMI. In all three case reports,

correlating laboratory findings with conventional autopsy observations enhanced the specific accuracy and

reliability that are functional in estimating postmortem interval.

Conclusion: Concomitant to the post-mortem interval, both biochemical and molecular markers are

complementary to routine forensic methods. Integration of biomarkers with autopsy findings,

histopathology, toxicology and scene investigation provides a multidisciplinary approach to increase the

credibility of medicolegal opinions. Extensive studies on biomarkers which are emerging with more

applications of forensic use but require to be decompressed in large-scale studies and repeatable, simple

protocols within routine forensic practice.

Keywords: Postmortem interval; Time since death; Forensic autopsy; Vitreous humour; Biomarkers;

Potassium; Hypoxanthine; Forensic pathology.

Introduction

One of the most important roles but also one of the hardest to give an exact answer in forensic pathology is

accurate estimation of postmortem interval (PMI) or Time Since Death (TSD). The PMI is important as it

can help reconstruct the events leading up to and surrounding death, corroborate witness statements, analyze

suspect alibis, and support medicolegal investigations. Common techniques that are performed as a standard

but are widely influenced by factors such as ambient conditions, the characteristics of the deceased

individual, the cause of death, and necrobiotic parks [1,2], making them unreliable especially during

intermediate and late postmortem interval periods [3].

Advances in forensic science have highlighted the importance of biochemical and molecular markers in

avoiding subjectivity and bias in PMI estimation. After death, the natural processes of cellular autolysis

CASE SERIES

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and tissue breakdown lead to familiar biochemical alterations in body fluids and tissues. Vitreous humour

is considered to be one of the more stable specimens because of its anatomic protection and delayed

postmortem degradation. Biomarkers including potassium in the vitreous humor, hypoxanthine, lactate,

sodium, chloride, urea and creatinine have shown strong positive correlations with PMI increasing overtime

and are increasingly being used in forensic investigations [3,4].

Moreover, molecular signatures such as RNA degradation profiles, microRNAs, inflammatory cytokines,

hypoxia-inducible factor-1 alpha (HIF-1α), aquaporins and other apoptosis-related proteins have been

proved to be of prospective value especially in the cases of decomposed bodies in which conventional

postmortem signs become less reliable [5–8]. Yet, interpretation of biopmarkers can be confounded by

premortem disease, environmental factors, agonal events and laboratory methodology so that one must

correlate these data with the findings during autopsy and scene investigation.

A considerable body of evidence has increasingly recommended chronicling biochemical, molecular,

histopathological, toxicological and customary autopsy conclusions to contemplate PMI in an incorporated

way. This illustrative three-case series illustrates the utilization of selected biomarkers over distinct

postmortem periods, showcasing their diagnostic merit and potential pitfalls while firmly establishing their

use as an adjunct in modern forensic practice.

Case Presentation

Case 1

Patient Information

A 38-year-old male was found unresponsive in his residence after reportedly being last seen alive

approximately 8 hours earlier. The deceased had no documented chronic medical illnesses. According to

family members, he had complained of mild chest discomfort the previous evening but did not seek medical

attention. There was no history of trauma, poisoning, or substance abuse.

Clinical History

The body was recovered from an indoor environment maintained at approximately 24°C. There was no

evidence of disturbance at the scene. Police investigation revealed no signs of foul play. The estimated

postmortem interval based on scene findings was between 6 and 8 hours.

Examination Findings

External examination demonstrated early rigor mortis involving the jaw and upper limbs, fixed posterior

lividity, and mild corneal clouding. No external injuries were identified. Internal examination revealed

moderate coronary artery atherosclerosis with acute thrombotic occlusion of the left anterior descending

coronary artery. The myocardium showed features consistent with an acute myocardial infarction.

Investigations

Vitreous humor analysis demonstrated elevated potassium and hypoxanthine concentrations consistent with

an early postmortem interval. Serum inflammatory biomarkers showed minimal elevation.

Histopathological examination confirmed acute myocardial ischemia. Toxicological screening was

negative for alcohol, illicit drugs, and common poisons.

Diagnosis

Sudden cardiac death secondary to acute myocardial infarction.

Treatment/Management

Not applicable, as the individual was declared deceased at the scene. A complete medicolegal autopsy with

biochemical, histopathological, and toxicological investigations was performed.

Outcome

Correlation of autopsy findings with vitreous biochemical markers supported an estimated postmortem

interval of approximately 7 hours, consistent with investigative findings.

Follow-up

The final autopsy report was submitted to the investigating authority, and the cause and estimated time

since death were accepted without discrepancy.

Case 2

Patient Information

A 56-year-old female was discovered deceased inside her residence after neighbours reported an unusual

Thivya R et al | DOI: 10.65188/nurexus.1081

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odour and inability to contact her for approximately one day. She had a documented history of type 2

diabetes mellitus and systemic hypertension.

Clinical History

The body was found indoors with no evidence of forced entry or physical struggle. Environmental

conditions were warm and humid. The estimated postmortem interval at the scene ranged between 18 and

24 hours.

Examination Findings

External examination demonstrated generalized rigor mortis, well-established lividity, and early abdominal

green discoloration. Internal examination revealed severe coronary atherosclerosis without evidence of

external trauma. Mild pulmonary oedema was present.

Investigations

Vitreous humor analysis revealed further elevation of potassium and hypoxanthine levels compared with

values expected during the early postmortem period. Lactate concentration was also increased.

Histopathological examination supported ischemic heart disease, while toxicological analysis did not

identify intoxicating substances.

Diagnosis

Natural death due to ischemic heart disease.

Treatment/Management

A comprehensive forensic autopsy with biochemical, toxicological, and histopathological investigations

was undertaken to establish the cause of death and estimate the postmortem interval.

Outcome

The combined interpretation of postmortem biochemical markers and classical autopsy findings indicated

a postmortem interval of approximately 22 hours, which correlated with the investigative timeline.

Follow-up

The medicolegal investigation concluded that the death resulted from natural disease, and no further

criminal inquiry was required.

Case 3

Patient Information

A 44-year-old male was recovered from an isolated agricultural field approximately three days after he had

been reported missing. No significant past medical history was available.

Clinical History

The body had been exposed to outdoor environmental conditions characterized by high daytime

temperatures and moderate humidity. Initial assessment suggested an advanced postmortem interval.

Examination Findings

External examination demonstrated moderate decomposition with abdominal bloating, skin slippage,

marbling, and insect activity. Conventional indicators alone were insufficient for precise estimation of the

postmortem interval. Internal examination did not identify fatal traumatic injuries.

Investigations

Biochemical analysis of vitreous humor remained feasible despite decomposition and demonstrated

markedly elevated potassium and hypoxanthine concentrations. Molecular assessment showed degradation

of selected RNA biomarkers, while immunohistochemical analysis demonstrated reduced expression of

viable tissue markers and increased apoptosis-related proteins. Toxicological analysis was negative.

Diagnosis

Undetermined natural death with moderate decomposition.

Treatment/Management

A multidisciplinary forensic evaluation incorporating autopsy findings, biochemical analysis, molecular

biomarkers, entomological assessment, and toxicology was performed.

Outcome

The integration of biomarker analysis with decomposition findings supported an estimated postmortem

interval of approximately 60 hours, which was consistent with police investigation and witness statements.

Follow-up

The comprehensive forensic report enabled successful reconstruction of the timeline of death and

contributed to the closure of the medicolegal investigation.

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Result

Table 1. Demographic and Case Characteristics of the Three Illustrative Forensic Autopsy Cases

Characteristic

Case 1

Case 2

Case 3

Age (years)

38

56

44

Sex

Male

Female

Male

Place of Body Recovery

Residence

Agricultural field

Estimated Postmortem

Interval (PMI)

7 hours

22 hours

60 hours

Environmental Conditions

Indoor (24°C)

Indoor (Warm and

humid)

Outdoor (Hot and

humid)

State of Preservation

Fresh body

Early decomposition

Moderate

decomposition

Suspected Cause of Death

Acute myocardial

infarction

Ischemic heart

disease

Undetermined natural

death

External Injuries

None

Autopsy Performed

Yes

Table 2. Summary of Autopsy and Laboratory Investigations

Investigation

Case 1

Case 2

Case 3

External Examination

Early rigor mortis,

fixed lividity

Generalized rigor mortis,

early decomposition

Skin slippage,

marbling, bloating

Internal Examination

Coronary artery

thrombosis

Severe coronary

atherosclerosis

No fatal traumatic

lesions

Histopathology

Acute myocardial

ischemia

Chronic ischemic heart

disease

Autolytic tissue

changes

Vitreous Humor

Analysis

Performed

Toxicology

Negative

Immunohistochemistry

Not indicated

Limited

Performed

Molecular Biomarkers

Not performed

RNA degradation

analysis

Investigation

Case 1

Case 2

Case 3

External Examination

Early rigor mortis,

fixed lividity

Generalized rigor mortis,

early decomposition

Skin slippage,

marbling, bloating

Table 3. Comparison of Biomarkers Used for Estimation of Postmortem Interval

Biomarker

Case 1 (Early

PMI)

Case 2

(Intermediate

PMI)

Case 3 (Late

PMI)

Forensic Significance

Vitreous

Potassium

Mildly

elevated

Moderately

elevated

Markedly

elevated

Reliable indicator of

increasing PMI

Vitreous

Hypoxanthine

Mild increase

Moderate increase

High

concentration

Reflects postmortem

cellular metabolism

Lactate

Slight

elevation

Moderate elevation

High level

Indicates progressive

anaerobic metabolism

Sodium

Mild reduction

Moderate reduction

Marked

reduction

Supports biochemical

estimation of PMI

Chloride

Stable

Mild decline

Moderate

decline

Adjunctive marker

Urea

Within

expected range

Mild elevation

Variable

Supportive biochemical

marker

Creatinine

Within

expected range

Mild elevation

Variable

Helpful when interpreted

with other markers

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IL-6

Minimal

expression

Moderate

expression

Increased

expression

Inflammatory response

after death

TNF-α

Low

Moderate

High

Associated with tissue

degradation

RNA Integrity

Preserved

Partial degradation

Marked

degradation

Useful molecular

indicator of PMI

Table 4. Timeline of Events in the Three Illustrative Cases

Event

Case 1

Case 2

Case 3

Last Seen Alive

Approximately 24

hours before discovery

Three days before recovery

Body Discovery

Morning

Following day

Third day after disappearance

Scene Examination

Completed

Medicolegal

Autopsy

Same day

Biomarker Analysis

Vitreous

biochemistry

Vitreous biochemistry

Vitreous biochemistry,

immunohistochemistry, molecular

analysis

Histopathology

Completed

Toxicology

Negative

Final PMI

Determination

7 hours

22 hours

60 hours

Final Forensic

Opinion Issued

Yes

Discussion

Estimation of the postmortem interval (PMI) is still a big challenge in forensic pathology due to

external/near surroundings, mode of death and biological factors affecting lifeless body after death.

While traditional methods (such as algor mortis, rigor mortis, livor morits and decomposition) are still

essential even today, their reliability becomes poorer with longer PMIs. Thus, biochemical and

molecular biomarkers have been developed as complementary tools to enhance the precision of PMI

estimation.

Owing to the anatomical isolation as well as propensity for resistance against postmortem deterioration,

vitreous humour exhibits some of the highest reliability in samples obtainable from postmortem

biochemical analysis. Donaldson et al. It has been reported that the use of biochemical analysis

performed on the vitreous humor is an accurate technique for determining PMI during the first 72 hours

after death [9]. Similarly, Focardi et al. In [10], the authors showed that the addition of other biochemical

markers to vitreous potassium contributes to the accuracy of estimation of PMI versus potassium alone.

In the current series of cases, increasing potassium concentration in vitreous correlated with increasingly

prolonged postmortem intervals, confirming previous studies.

With the recent finding that hypoxanthine is overproduced in cell culture after stopping metabolism,

this metabolite has also emerged as an important biomarker since it accumulates only when cellular

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metabolic turnover ceases. Zilg et al. Aramani et al. [11] showed a significant relationship between

vitreous hypoxanthine and postmortem interval (PMI). Combination assessment of potassium and

hypoxanthine improves diagnostic performance. [12] Here we show that progressive postmortem

biomarkers changes are similar across postmortem interval in our cases.

Progress on the field of forensic molecular pathology has also recently bolstered PMI (Post Mortem

Interval) estimation. Palacio et al. Maiese et al. [13] also shed light on the importance of electrolyte

analysis in advanced forms, The RNA degradation profiles and the other immunohistochemical markers

were preliminarily showed by [14] that these might further help towards the diagnosis, especially for

decomposed bodies. In agreement with these observations, molecular biomarker analysis in the third

case supplemented standard autopsy findings and provided more accurate estimation of postmortem

interval.

Pathophysiology

In the post-mortem period, complete discontinuation of circulatory function leads to hypoxic conditions

in the tissues, resulting in depletion of ATP, failure of sodium-potassium pumps and gradual process

called cellular autolysis. These changes lead to an efflux of intracellular potassium into the extracellular

compartment, producing a gradual rise in vitreous potassium level. Meantime, adenosine nucleotides

are degraded to hypoxanthine and anaerobic metabolism leads to lactate accumulation. Decomposition

occurs in a stepwise manner, involving the degradation of proteins and nucleic acids, activation of

inflammatory pathways, and progressive fragmentation of RNA that can be quantified as molecular

shifts consistent with elapsed postmortem time. These biochemical and molecular events can be

trivialized to elucidate the sequential proteomic alterations in these three representative cases.

Clinical Significance

Estimating the postmortem interval (PMI) is a decisive criterion for reconstructing the events between

death and corpse discovery, validating witness evidence, assessing suspect alibis, and aiding judicial

processes. Biomarker analysis provides objective laboratory findings that serve as a complement to

autopsy results, thereby improving the scientific reliability of forensic opinions. This case series

highlights the best use of vitreous biochemical markers, molecular investigations and routine autopsy

findings in a complementary manner to enhance the estimate of PMI, especially for cases with

decomposed bodies or doubtful spacing.

Diagnostic Challenges

Despite the promising progress, there are few disadvantages associated with biomarker-assisted PMI

estimation. Factors such as premortem diseases, agonal events (thermal status), ambient temperature

and humidity at the time of collection, microbial activity, handling and storage conditions before

analysis, as well as laboratory methodology have the potential to affect biomarker levels. Madea et al.

R [15] made it clear that no sole biochemical or molecular architecture can calculate PMI correctly

across all forensic situations.

Novelty of the Case Series

The current illustrative case series is novel in its demonstration of the application of multiple

postmortem biomarkers at both early, intermediate and late postmortem intervals in a single forensic

context. This case series integrates vitreous biochemical analysis, molecular biomarkers,

histopathological findings, toxicological evaluation and conventional autopsy observations unlike

reports focusing on individual biomarkers to demonstrate a multidisciplinary approach towards PMI

estimation. This amalgamates contemporary practice in forensic pathology and addresses an evolving

role of laboratory biomarkers within medicolegal investigations.

Strengths and Limitations

The main strength of this case series is the combination of biochemichal and molecular markers with

classics autopsy process, histopathology, toxicology and laboratory investigations in postmortem

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interval increase accuracy. Note that the cases are purely illustrative, for pedagogical purposes, and not

intended to be quantitative validation of biomarker performance. Differences in environmental

conditions, cause of death, and laboratory methods may impact levels of certain biomarkers. These

findings should be confirmed in multicentre studies employing standardized protocols so that these

biomarkers can be used routinely in forensic practice.

Summary

This three-case series of illustrative examples demonstrates the use of biochemical and molecular

biomarkers in estimating post-mortem interval across early, intermediate and late aspects after death. The

progressive changes in vitreous humour potassium, hypoxanthine and other laboratory parameters were in

agreement with the increasing postmortem intervals and corroborated conventional autopsy findings.

Histopathology, toxicological and molecular investigations combined with biochemical analysis also

enhance the reliability of estimating a postmortem interval especially in decomposed cases where

conventional methods may not lead to accurate estimation. The conclusions highlight that laboratory

biomarkers are an increasingly important adjunct for forensic pathologists but reinforce that their

interpretation should always be performed with the benefit of a complete medicolegal assessment.

Conclusion

Accurate estimation of postmortem interval (PMI) is essential to forensic pathology and medicolegal

investigations. While traditional postmortem changes are still useful, their reliability is affected by

environmental and biological factors. In this triad descriptive case series, it was evidenced how potassium

and hypoxanthine in vitreous humour may complement the statistics on PMI estimation with biochemical

and molecular biomarkers, respectively. Autopsy, histopathology, toxicology and scene investigation

integration with these biomarkers reflect more credibility than single entity approach in forensic

conclusions. This emphasizes the need for new large-scale studies and standardized protocols for future

forensic procedures as soon as emerging biomarkers become validated.

Declaration

Informed Consent: Written informed consent for publication should be obtained from the legally

authorized representative or next of kin before publication of actual forensic case reports or case series. In

the present educational manuscript, no real patient identifiers or identifiable clinical information have been

used.

Conflict of Interest: The authors declare that they have no competing interests or conflicts of interest

related to this work.

Funding: Nil

Authors' Contributions: All authors contributed substantially to the conceptualization, literature review,

manuscript drafting, critical revision, and final approval of the manuscript.

Acknowledgements: The authors acknowledge the valuable contributions of forensic medicine

professionals and researchers whose work has advanced the understanding of postmortem biomarkers and

their applications in medicolegal investigations.

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