The time since death or post-mortem interval (PMI) is the time interval from the death of an individual to the time of examination.  In medico-legal autopsies it is very important to give a reliable time-frame since death as it can help include or exclude the suspects involved in the crime, especially when no witness is present. An erroneous PMI can lead the investigation into a wrong path. Unfortunately, no definite or precise method which can fairly accurately estimate the PMI has been devised till now.

The traditional methods of estimation of the PMI are based on post-mortem physical changes, like rigor mortis, livor mortis, algor mortis and corneal haziness. These physical changes are highly influenced by various external as well as internal factors. So, these methods are now considered unreliable, or they give a wide range of time frame that is impractical in its use in the investigation of crime.

So, the recent studies of the estimation of the PMI are based on chemical changes that occur in the body after death, on thanatochemistry. Post-mortem chemical analysis can be helpful in:

  • Estimating the time since death,
  • Finding the cause of death, and in
  • Toxicological analysis.

Since 1960, many studies have been carried out to estimate the time since death by chemical analysis. The various analytes that can be studied for the PMI estimation are:

  • Potassium, Sodium, Chlorides,
  • Hypoxanthine, Xanthine, Lactates,
  • Glucose, and
  • Amino acids.

The study of these analytes are based on the principles that after death there is a rise or fall in the concentration of the analytes,  and that this is time-dependant. This rise or fall is because of the following reasons:

Electrolyte (Sodium, Potassium and Chlorides):

Dysfunction of the Na-K ATPase pump:  After death, there is depletion of ATP, hence, the ATP-dependant pumps begins to fail. So, the concentration gradient of sodium and potassium which is maintained by the Na-K ATPase pump by the active transport of the electrolyte against its concentration gradient is lost.

Autolysis: Autolysis is the aseptic enzymatic breakdown of body tissue. After the breakdown of the cell membrane, the selective permeability that maintains the concentration difference inside and outside the cell is lost. This is the Fick’s law of diffusion.

So, there will be diffusion of electrolytes along its concentration gradient. Potassium, being in high concentration in the intracellular compartment, will diffuse out into the extracellular fluid. Sodium and chloride, being in higher concentration in the extracellular fluid, diffuse into the intracellular compartment. So, with an increase in time, there is a gradual increase of potassium and a fall in sodium and chloride concentrations in the body fluid.

Metabolites (Hypoxanthine, Xanthine, Lactate):

Metabolic activity doesn’t cease immediately after death. Anaerobic metabolism continues for some time after death, so the product of metabolism can also be studied to estimate the time since death. There is a  progressive fall in the glucose and a rise in the lactate level in the body fluid.

The oxypurines, hypoxanthine, xanthine and uric acid are the terminal products of purine catabolism in man. Hypoxanthine is also a biochemical marker of hypoxia that accumulates in body fluids during hypoxic degradation of adenosine monophosphate (AMP) within minutes after a hypoxic event.

Product of protein breakdown:

There is an increase in amino acids due to protein breakdown.

Other products of bacterial metabolism:

Among these analytes, the most extensive studies have been done on potassium and hypoxanthine.

Biological Samples that can be used are:

  • Blood ,
  • CSF,
  • Vitreous humor,
  • Synovial fluid, and
  • Pericardial fluid.

Among these body fluids, before 1960, more studies were carried out on blood and CSF.  But it was soon realized that the semi-permeability of the cell membrane in these fluid is lost soon after death. Hence, these fluids are now not used for estimation of the PMI.

The concentration gradient after the loss of selective permeability is lost after a few hours in blood, after 15 to 20 hours in CSF and after 120 hours in vitreous humor.

Ideal fluid/sample

The ideal sample for chemical analysis for the estimation of the PMI should be stable, inert, easy to obtain, well-protected from bacterial contamination, and should have a slow rate of decomposition.

Vitreous humor

Vitreous humor fulfils almost all the criteria for an ideal fluid; so, after 1960, more studies were based on vitreous humor. Vitreous humor is an inert, colorless, viscous, gel-like substance which is present in the posterior chamber of the eye ball which is bound anteriorly by the lens and cilliary body and posteriorly by the retina. It is around 4 ml in volume, which is approximately two-thirds of the entire globe [1, 3]. Vitreous humor is composed of 99 % water, collagen fibers, hyaluronic acid, soluble protein, sugar, ascorbic acid, lactic acid, and electrolytes.

The first study on vitreous humor was done by Naumann NH in 1959. In 1963, Sturner first studied vitreous potassium for the estimation the PMI.

Vitreous humor is collected  with a 5 ml syringe and an 18 gauge needle by a sclera puncture made as much laterally as possible by retracting the lateral canthus. The needle is directed towards the centre of the globe with an angulation of 60 degrees. Around 1.5 to 2 ml of vitreous humor is collected by gentle and slow traction on the syringe. To maintain the contour of the globe, the  same amount of normal saline should be replaced into the globe

Pre-analytical procedure:

Since vitreous humor is viscous, it can pose difficulty in pipetting during the analytical procedure. Thus, a pre-analytical treatment is used to reduce its viscosity. Various pre-analytical procedures are available, but the simplest method is to centrifuge it at 3,500 rpm for five minutes.

The formulae for the estimation of the PMI obtained by the various authors are as follows:

Author/year Formula obtained
Sturner (1963) PMI (hours) = 7.14 [K+ ] – 39.1
Madea et. al. (1989) PMI (hours) = 5.26 [K+ ] – 30.9
James et. al. (1997) PMI (hours) = 4.32 [K+ ] – 18.35

 

Synovial fluid:

Synovial fluid is a colorless viscous fluid present in all synovial joints of the body that helps lubricate the joint. It is well-protected by the synovial membrane of the joint. It is secreted by the cells lining the synovial membrane. It is a derivative of plasma with a higher amount of hyaluronic acid

It is widely used in clinical rheumatology.

Before 2001, it was studied for the estimation of alcohol concentration, drug distribution and cause of death. In 2001, Madae B studied the PMI using synovial fluid and found that the accuracy is similar to that of vitreous humor.

Thus, synovial fluid can be used in mutilated bodies, when the head or eyes are missing and in cases of ocular trauma.

Drawbacks:

  • No reference values.
  • Lack of standardized pre-analytical procedures.
  • Lack of a standardized analytical procedure. The values differ according to methods applied.
  • The instruments used are validated and calibrated for the use of blood and urine.
  • Poor reproducibility.

Recent advances

H magnetic resonance spectroscopy (H MRS)

Once putrefaction is apparent, estimation of the PMI becomes even more difficult and unreliable.

Thirty years ago, protein degradation products in the brain were studied and a method of estimation of the PMI on the basis of amino acid concentration in the brain was proposed.

Recently, magnetic resonance imaging (MRI) is being used to identify protein degradation metabolites released in the brain during decomposition. First, the metabolites have to be characterized. About 30 such metabolites have been identified and 19 of them show well-defined time courses. The study is being done on sheep, pigs and on selected human brains. The animal and human brain showed similar metabolites at similar time courses. Five metabolites studied can give a relatively accurate PMI of up to 250 hours and 10 metabolites up to 400 hours. This is a non-invasive chemical analysis.

This research is currently being carried out by a group of researchers at the Institute of Forensic Medicine and the Department of Clinical Research, University of Bern, Switzerland.

 

Immunohistochemistry

The process of detecting antigens (e.g., proteins) in the cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues is immunohistochemistry.

Detection of insulin, thyroglobulin and calcitonin: The theory behind this is that these compounds are hormones, i.e., functional proteins, and that  with the increase in the PMI, the structures of these proteins degrades and denatures and immunohistochemical staining becomes negative with time. Thyroglobulin in the thyroid stains positive for five days post-mortem and negative after 13 days. Insulin in the B-cells of the pancreas stains positive for 12 days and negative after 30 days post-mortem and  calcitonin in the C-cells of thyroid positive up to four days and negative after 13 days post-mortem.

DNA degradation

Recent studies on DNA degradation for the prediction of the post-mortem interval is by flow cytometric evaluation. Flow cytometry is a laser- or impedance-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending the cells in a stream of fluid and passing them through an electronic detection apparatus. It can also detect DNA degradation.

At first DNA degradation was thought to be independent of any external factors. But since DNA can also be degraded by decomposition, it has now been proved that this method is also influenced by various external as well as internal factors. So, again, the accuracy of this method is not reliable.

So, researchers are still in search of a reliable new method which can more accurately estimate the time since death.

References

  • Madae B, Kreurer C, Banashhak S. Postmortem biochemical examination of synovial fluid — a preliminary study. Forensic Science International April 15 2001; 118(1):29-35.
  • Prasad B K, Choudhary A, Sinha J N. A study of correlation between vitreous potassium level and postmortem interval. Kathmandu University Medical Journal. 2003; 1(2): 132-134.
  • Bohra B et. al. Estimation of Postmortem Interval by Measuring Potassium Level in Vitreous Humor. Journal of Indian Academy of  Forensic Medicine. October-December 2014; 36(4):374-8.
  • Sachdeva N, Rani Y, Singh R, Murari A. Estimation of Post-Mortem Interval from the Changes in Vitreous Biochemistry. Journal of Indian Academy of Forensic Medicine. April-June 2011; 33(2): 171-4.
  • Salam H, Shaat E, Hassan M, MoneimSheta A, Hussein H. Estimation of postmortem interval using thanatochemistry and postmortem changes. Alexandria Journal of Medicine. 2012; 48(4): 335-44.
  • Ahi R, Garg V. Role of Vitreous Potassium level in estimating Postmortem Interval and the factors affecting it. Journal of clinical and diagnostic research. 2011 Feb; 5(1):13-15.
  • Tumram N K, Ambade V N, Dongre A P. Thanatochemistry: Study of vitreous humor potassium. Alexandria Journal of Medicine. 2014; 50: 365-368.
  • Paranitharan P, Pollanen M S. Utility of postmortem vitreous biochemistry. Sri Lanka Journal of Forensic Medicine, Science & Law. May 2011; 2 (1): 23-25.
  • Kumar A, Singh A, Harish D, Chavali K H. Use of vitreous humor in comparison to use of routine viscera for chemical analysis in suspected poisoning case. Journal of Punjab Academy of  Forensic Medicine and Toxicology. 2011; 11(2): 72-76.
  • Madea B, Musshoff F. Postmortem Biochemistry. Forensic Science International. 2007; 165: 165-171.
  • Millo T, Jaiswa A K, Behera C. Collection, preservation and forwarding of biological samples for toxicological analysis in medicolegal casas: A review. Journal of Indian Academy of Forensic Medicine.2008; 30(2): 96-100.
  • Tumram N K, Ambade V N, Dongre A P. Thanatochemistry: Study of Synovial fluid Potassium. Alexendria Journal of Medicine. 2014; 50:369-372.
  • Botezatu G A. An examination of the time of death based on the rectal temperature data, the biochemical indices of the blood & the pericardial fluid. Sud Med ekspert (English version) Moscow. 1977; 20(1):39-43.
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