Methods Of Estimation Of Time Since Death

Introduction

• Forensic pathologists estimate Time Since Death (TSD) to:
  • Assist in death investigations.
  • Limit the investigation period.
  • Narrow down the number of suspects in homicide cases.
  • Establish the timeline of events leading to death.
• Numerous methods exist, but all are affected by variable factors.
• Authors advocate using multiple qualitative and quantitative systems for reliability.

Function: Post-Mortem Interval (PMI)

• Traditionally classified into three stages:
  • Immediate
  • Early
  • Late

Immediate PMI (0-3 hours)

• Characterized by rapid biochemical and physiological changes due to:
  • Absence of blood circulation.
  • Loss of regulatory mechanisms.
• Changes primarily detectable in eyes and skin.
• Eye Changes:
  • Trucking: Segmentation of retinal blood vessels.
    • Appears as a break in the blood column during ophthalmoscopic examination.
    • Occurs within 0.5 to 2 hours after death.
  • Loss of intraocular pressure:
    • Decreases drastically, reaching 4 mmHg or less within 6 hours.
  • Clouding of the cornea:
    • Begins within 2 hours, preventing intra-ocular examination with an ophthalmoscope.
• Skin Changes:
  • Loss of elasticity and luster within the first few hours.
  • Skin appears pale.
  • No morphological changes observed in histological examination within 6 hours PMI.
• Other Examinations:
  • Lack of cellular or biochemical changes within 3 to 6 hours post-mortem.
• Emptying of Gastric Contents:
  • Small light meals empty within 1 to 3 hours.
  • Time of consumption, volume, and type of meal can help estimate PMI.
• The immediate post-mortem phase is the interval between somatic and cellular death (2-3 hours).
  • Usually denotes a lack of discernible changes in morphology or histo-chemistry.

Early PMI (3-72 hours)

• Most important period for PMI estimation in medico-legal cases.
• Estimation of TSD is most relevant for establishing the timeline of events.
• Estimated using the classical triad of post-mortem changes:
  • Rigor mortis
  • Livor mortis
  • Algor mortis

Algor Mortis

• Cooling of the body after death due to:
  • Loss of homeostatic regulation by the hypothalamus.
  • Heat loss to the environment via conduction, convection, and radiation.
• Most accurate method of estimating TSD in the early post-mortem phase.
• Requires intensive knowledge and research due to numerous influencing factors.
  • Temperature gradient between body and ambient temperature.
  • Differences in temperatures of different localities.
• Rule of thumb: Decrease of $1.5$ degrees Fahrenheit every hour.
• Several charts, formulae, and algorithms have been developed
  • Henssge’s nomogram is the most widely taught.
• TSD estimation involves measuring rectal temperatures.
• Nomograms for brain-temperatures have also been developed.
  • Brinkmann et al. (1976, 1978)
  • Henssge et al. (1984)

Rigor Mortis

• Post-mortem stiffening of muscles caused by depletion of adenosine triphosphate (ATP).
  • ATP is required for the breakdown of actin-myosin filaments in muscle fibers.
  • Actin and myosin form covalent bonds during contraction.
  • Cessation of oxygen supply stops aerobic respiration, leading to lack of ATP production.
• Rigor mortis starts immediately after death and follows a sequence: "march of rigor" or Nysten’s Law.
• Develops simultaneously in all muscle tissue, but perceptibility varies.
• Smaller muscles (face) are affected first, followed by hands, upper limbs, and lower limbs.
• Appearance:
  • Begins approximately 2 hours after death in the face muscles.
  • Progresses to the limbs over the next few hours.
  • Completes between 6 to 8 hours after death.
• Stays for another 12 hours (until 24 hours after death).
• Disappearance:
  • Actin-myosin complex disintegrates due to proteolysis.
  • Begins in smaller muscles of the face, followed by upper limbs and lower limbs.
  • Generally disappears in 36 hours after death, followed by secondary flaccidity.

Livor Mortis

• Purplish-blue discoloration of the skin in dependent parts of the body.
• Caused by collection of blood in skin vessels due to gravitational pull.
• Development:
  • Hypostasis develops as spots of discoloration within 0.5 to 2 hours.
  • Spots coalesce to form larger patches.
  • Forms uniform discoloration of dependent parts not subject to pressure, appearing from 6 to 12 hours.
• Becomes ‘fixed’ after a certain period due to disintegration of blood cells and seepage of hemoglobin.
• Fixation is confirmed by applying pressure with thumbs and traditionally indicates a PMI greater than 12 hours.
• Colorimetric methods have been developed for objective PMI estimation.

Histo-morphological Analysis

• Total and differential blood counts, and microscopic examination of blood, are used for TSD estimation.
• All blood cells were not identifiable beyond 84 hours after death.
• Blood cell counts decrease beyond 84 hours after death.
• Histological studies of the skin:
  • Degenerative changes appear 6 hours after death (vacuolation of corpus basale and spinosum).
  • Dermo-epidermal separation is seen 9 hours after death.
  • Dermis shows rarefaction and disintegration 6 and 18 hours after death, respectively.
• Glycogen depletion:
  • Glycogen in the basal membrane of sweat glands, secretory cells cytoplasm, and duct cells depletes within 3 hours PMI, leading to PAS-negative cells on histology.
  • Basal membrane continues to show magenta staining up to 18 hours post-mortem.
• Eccrine sweat glands show vacuolation after 3 to 4 hours of PMI, and cells disintegrate completely 15 hours after death.
• Sebaceous glands appear normal until 18 hours post-mortem, followed by separation of layers and disintegration of hair papilla.
• Pleocytosis can be used to estimate the PMI using a third-order polynomial equation.
  • Cells are primarily lymphocytes with a significant fraction of macrophages, which become vacuolated and unidentifiable after 12 hours.

Biochemical Assessment

• Blood assessment is non-significant in the immediate post-mortem phase due to lack of cellular death.
• Cellular death in the early phase makes biochemical blood assessment difficult.
• Electrolytes redistribute from cells into plasma and serum, causing varying changes in electrolyte levels (studied in thanato-chemistry).
• Assessment useful for PMI estimation from:
  • Vitreous humor
  • Synovial fluid
  • Pericardial fluid
  • Urine
  • Cerebrospinal fluid
• Numerous factors need to be accounted for:
  • Age
  • Gender
  • Biological background
  • Lifestyle
  • Cause of death
  • Intrinsic and extrinsic factors
• Only a few biochemical markers have had sufficient investigation:
  • Potassium
  • Sodium
  • Urea
  • Chloride
  • Magnesium
  • Hypoxanthine
  • Cardiac troponin T
• Assessment for their potential use revealed:
  • 0 markers suitable for use.
  • 6 markers suitably researched but not suitable for practical use.
  • 18 markers poorly investigated and not suitable for application.
  • 364 markers with insufficient information.

Supra-vital Reactions

• Proposed as a means of estimation of PMI.
• Madea defines PMI into four stages:
  • Latency period: Tissue performs aerobic respiration until depletion of stores.
  • Survival period: Loss of tissue function, but can be re-activated using external stimuli (e.g., electrical stimulation of nerves).
  • Resuscitation period: Ability of tissue to recover is completely lost.
  • Supra-vital period: Survival period of tissue after complete, irreversible ischemia.
• Survival period encompasses the latency period.
• Resuscitation period encompasses both the latency and survival periods.
• Supra-vitality period includes all the previous three.
• Supra-vitality differs from the resuscitation period in that the tissue is excitable irrespective of recovery of function.
• Example:
  • Skeletal muscle: resuscitation period $2$ to $3$ hours, supravital period up to $20$ hours.
  • Cardiac muscles: resuscitation period $3.5$ to $4$ min, supravital period up to $2$ hours.
• Method developed using electric excitability of Orbicularis oculi with surface electrodes.
  • Ratio of relaxation time and maximum force (force-related relaxation time) is reliable for estimating PMI.
• Super-sensitivity of tissue in the immediate post-mortem phase is called Zsako’s phenomenon.
• Supra-vital reaction examines idio-muscular or local contraction, not the entire muscle.

Late PMI

• Body tissue starts disintegrating.
• Describable as:
  • Decomposition or putrefaction
  • Adipocere formation
  • Mummification
  • Skeletonization
• Complex tissue breaks down into simpler molecular forms by:
  • Body’s enzymes
  • Bacteria in/colonizing the remains.
• Body primarily undergoes decomposition or putrefaction:
  • Greenish discoloration
  • Bloating due to gas formation
  • Liquefactive necrosis
• Decomposition depends on:
  • Climate
  • Season
  • Body weight
  • Clothing
• Decomposition can be divided into five stages:
  • Fresh
  • Early decomposition
  • Advanced decomposition
  • Skeletonization
  • Extreme decomposition

Fresh Phase

• Starts as early as 24 hours and as late as 7 days after death (especially in colder months).
• Shows no insect activity other than blow fly-eggs in cavities and areas of tissue dehiscence.
• Egg deposition can occur in living patients, especially immobile and debilitated subjects.

Early Decomposition Phase

• Begins with the onset of skin slippage and hair loss.
• Changes usually begin from the first day to up to five days post-mortem.
• Maggots begin to appear on the body from the second-day post-mortem.
• Body appears grayish-green with marbling present.
• Right iliac fossa is the first body part to show greenish discoloration (as early as the second-day post-mortem).
  • Due to the superficial position of the caecum.
• Extremities appear brownish with drying of the skin, especially over the fingers, nose, and ears (usually beginning on the second post-mortem day).
• Greenish discoloration progresses to the entire abdomen with concurrent bloating (may start on the second day).
• Bloating advances to the rest of the torso and subsequently the body, resulting in crepitations over the entire body on handling.
• Phase is also associated with purging (release of decomposition fluid from the orifices) and a strong disagreeable odor.
• Bloating may be seen as early as three days after death and subsides by the second-week post-mortem due to disruption of abdominal gases.
• The body appears blackish-green by the second week.
• The body appears brownish-black with the leathery appearance of skin.
  • Usually seen until the end of the first month but may be prolonged to two months.
• Underlying tissue also appears darkened with the texture changing to a viscous, slimy paste.
• Between the tenth day and the end of the first month, maggot activity continues under the leathery skin, with the skin further desiccating to form a hardened leathery shell, with loss of underlying soft tissue.

Advanced Decomposition Phase

• Begins with the appearance of loose sagging skin and the collapse of the abdominal cavity.
• Body shows extensive maggot infestation.
• Changes appear as early as the fourth-day post-mortem, but may begin as late as ten days after death.
• Loss of soft tissue, including the loss of the desiccated leathery skin, results in exposure of less than half of the skeletal material.
• This phase is usually associated with the presence of pupal cases, as well as the appearance of molds over the body and clothing.
  • Usually occurs in the second month but may occur six to nine months post-mortem.
• Desiccation of the outer skin could accompany the structural retention of internal organs, or be accompanied by autolysis and loss of internal organs.
• Decomposition may progress rapidly in buried remains or remains left in an environment with high humidity, resulting in extreme maggot activity, accelerated autolysis, and could progress directly to skeletonization or adipocere formation, without desiccation and mummification of the skin and outer tissue.
• Remains may undergo either saponification or desiccation.
  Adipocere Formation:
• Warm, humid, and oxygen-lacking environments favor the development of adipocere.
• Adipocere is a waxy substance that results from anaerobic bacterial hydrolysis of body fat.
• Primary organism responsible is Clostridium perfringens, causing aggregation of crystal of fatty acid, resulting in loss of cellular detail as well as the loss of epidermis.
• Formation time depends on pH, temperature, moisture, and lack of oxygen.

Skeletonization Phase

• Results in the exposure of more than half of the skeletal elements, which could still demonstrate soft tissue is still attached.
• Usually associated with desiccated tissue or adipocere over less than half of the body.
• Desiccated tissue most commonly appears at muscular or ligamental attachments along the vertebral column as well as ends of long bones.
• Adipocere is commonly seen over the thighs with high subcutaneous fat deposits.
• This stage appears two months after death, although it usually presents between two and nine months post-mortem.
• Continuation of decomposition leads to exposure of all osseous material, with only some greasy material left behind or exposing dry bones.
  • Usually seen after six months of exposure, although it has been reported to have occurred as early as the third week.
• This stage can last for years if the elements are not exposed, as is seen in buried remains or remains found indoors.

Extreme Decomposition Phase

• Seen only in remains exposed to the environment.
• Leads to erosion of the skeletal elements.
• Begins with the process of bleaching of bones.
  • Commonly seen six months after exposure, although it has been documented to appear as early as two months after death and as late as two and a half years post-mortem.
• Skeletal elements undergo further degeneration of the cortical structure, resulting in a metaphyseal loss in long bones and exposure of the cancellous part of spongy bones.
  • Seen commonly between a year to a year and a half after death, although it has been reported to have occurred as early as the fourth month.
• The metaphyseal loss was reported to have occurred at PMI of five and a half years.

Forensic Entomology

• Routine practice for PMI estimation in early and late post-mortem periods.
• Two methods of estimation:
  • Succession-based approach
  • Development-based approach

Succession-Based Approach

• A suitable succession-model is chosen for use, one that corresponds to the environmental conditions, including the circumstances of death.
• Research needed into the effect of environmental factors on decomposition and insect succession.
• Mañas-Jordá demonstrated that different taxa were found to be prevalent based on environmental conditions.
  • Species diversity and individual numbers were examined.
  • No species association with Stage I and II of decomposition.
  • Three species [Compsomyiops spp (Diptera: Calliphoridae) and C. irazuana, and Megagrapha sp1 (Hybotidae)] associated with Stage III.
  • Two species [Mesosphaerocera sp1. and Fannia sp1. (Fanniidae)] associated Stage IV.
  • One species [Stilpon sp1 (Hybotidae)] associated with Stage V at Huitepec Natural Reserve.
  • At the City of San Cristóbal de las Casas:
    • Four species [Prochyliza brevicornis (Melander; Diptera: Piophilidae), C. latifrons, Lucilia mexicana (Calliphoridae) and Compsomyiops spp (Macquart)] associated with Stage II.
    • Three species [Synthesiomyia nudiseta (Vanderwulp), Musca sp1, and Hydrotaea sp1(Diptera)] associated with Stage III.
    • One species Chrysomya rufifacies (Macquart; Diptera: Calliphoridae) and Fannia sp1 associated with Stage IV.
    • Stilpon sp1 was found to be associated with Stage V.

Development-Based Approach

• Looks at the presence of different stages of the insect on the body and in the surrounding area to help estimate PMI.
• Matuszewski used L. caesar (Diptera: Calliphoridae), Thanatophilus sinuatus, and N. littoralis (Coleoptera: Silphidae) to demonstrate that the presence of a developmental stage and absence of the subsequent developmental stages of carrion insects, could be used in conjunction with the estimation of their pre-appearance interval (PAI) to develop a reliable estimation of PMI.
• It is, therefore, essential to establish known PAI values for different insects in the environment under examination.

Molecular Assessment

• Recent advances have led to various advances in the estimation of PMI.
• The degeneration of mRNA, DNA, and proteins are evaluated and can be used to estimate the PMI.
• RNA transcripts were found to be the most relevant due to their rapid degeneration and temporal correlation.
• Multiple studies demonstrated a linear correlation between PMI and degeneration.
• This correlation was found to be temperature and tissue-dependent.
• A study from Porto, Portugal examined 11 gene transcripts for correlation with TSD.
  • 8 murine tissues were divided into three groups:
    • Group I: heart, spleen and lung.
    • Group II: femoral quadriceps, liver and stomach.
    • Group III: Pancreas and skin.
  • Samples from groups I and II were serially analyzed.
  • RNA degeneration was time-dependent for the entire 11 hours, although no statistical significance was demonstrable for the first four hours.
  • Researchers selected 11 genes for quantitative PCR analysis.
  • While RNA in the heart was found to be most stable, it showed no correlation with PMI.
  • A total of six genes were found to correlate with PMI:
    • Four in the femoral quadriceps (Actb, Gapdh, Ppia, and Srp72).
    • Two genes in the liver (Alb and Cyp2E1).
  • Mathematical models were developed to estimate PMI with an error mean of 51.4 minutes.

Issues of Concern

• Numerous factors beyond the scope and control of research affect the rate of post-mortem changes in real-life scenarios.
• Requires the use of multiple methods of estimating PMI to help establish a reliable timeline.
• Complicated by slight variations in sample collection and processing.
• Individual variations in:
  • Temperatures at time of death (body and ambient).
  • Clothing worn by the deceased.
  • Circumstances of death (including cause of death, preservation after death, exposure to the elements etc.).
  • Methods of retrieval.
  • Storage and preservation of samples before analysis.

Clinical Significance

• Information about time since death is an essential feature of postmortem reports issued by the forensic pathologists.
• Estimation of post-mortem interval provides useful information regarding the time of death, and hence, the same is argued upon by the defense in the court of law.
• Clinical expertise is warranted to ensure that the postmortem changes are well- interpreted and inferences get drawn correctly.
• The estimated time since death or the PMI may range in hours and sometimes in days when much time has elapsed after death, which may not always be helpful to law enforcement, who would require a narrower range of estimates to help in the investigation.
• The need for further research is, thus, emphasized.

Enhancing Healthcare Team Outcomes

• Estimating the time since death is among the pertinent challenges faced by forensic pathologists and law enforcement agencies all over the world.
• Requires collaborative efforts from an interprofessional team:
  • Bio-chemist
  • Pathologists
  • Anthropologists
  • Forensic pathologist
  • Law enforcement personnel
• Other health care practitioners such as clinicians and nursing need to have a basic understanding of the processes, so they can provide input when requested, and be aware not to damage any evidence accidentally.