The Value of Post-Mortem Biochemistry

F.J. Tames

What is Death ?(1)

The accepted legal definition of Death is the cessation of life in a previous living organism. Cellular death is the cessation of respiration and the normal metabolic activity within the body tissues and cells. This process can take 3 Minutes to 16 Hours. Somatic death means the individual will never again communicate or interact with the environment. The individual is irreversibly unconscious.

What is Death ?(2)

Human Body is a highly complex assembly of dynamic, but coordinated, chemical factories. Constant Energy expenditure required After Death.or a Strike !! Integrity and cooperation of the factories break down at surprisingly differing rates after death Concentration gradients, metabolism changes

Is Death therefore a process ?

Algor Mortis & Autolysis 4 mins-1 Hour Rigor Mortis or Cadaveric Rigidity 3-8 Hours Post-Mortem Hypostasis after 12 Hours Decomposition after 3-4 Days Putrefaction or Mummification after 2 Weeks Adipocere 3-8 Weeks Skeletalization after 2 Years

Useful Specimens for post mortem Biochemistry?

Ante Mortem Blood, Serum, Plasma etc. Post Mortem Blood Specimens Post Mortem Urine, Stomach Contents & Hair Post Mortem Liver Post Mortem Bile, Brain, Lungs, Kidney, Nasal Swabs Post Mortem Excised Tissue, Injection Sites Post Mortem Vitreous & Aqueous Humour

Sample Preparation for post mortem Biochemistry

DO NOT milk Blood from a Limb NO NOT send Cardiac Blood Use 2 peripheral sites if possible Centrifuge a portion of Blood A.S.A.P. VH from both eyes into SMALL tubes with Fluoride preservation of a portion Use the telephone BEFORE the scalpel !

Historical post mortem Biochemistry

1902 Specific Gravity of Blood from each side of the heart to demonstrate either fresh or sea water drowning. Blood on left side of heart noted as diluted in most fresh water drowning cases. 1960 Measurement of Blood Sodium, Chloride & Magnesium demonstrates sea water drowning. 1981 estimation of free Histamine and serotonin demonstrate time of death from gunshot wound

The Vitreous Humour(1)

Sterile medium for Biochemical analysis No Esterases or Bugs Reflects Ante Mortem Biochemical state VITAL to collect sample from BOTH eyes Fluoride preservation However, Small volume

The Vitreous Humour(2)

Usually, a clear, serous fluid easy to work with analytically using laboratory equipment Anatomically isolated & fenced fluid 99% Water with Type II Collagen, Hyaluronic Acid. Ultafiltrate. Turn Over 12-28 Hours. CAUTION. Body submersion in water leads to fluid diffusion out of the Vitreous Humour

Vitreous Humour Electrolytes

Pounder & Coe studied 2435 paired VH samples for electrolytes, Urea, Creatinine and others Sodium reported to fall, Chloride stable while Potassium & Bicarbonate rise in a time dependent manner post mortem. Glucose stable 6-12 months at 4oC Urea, Creatinine & Acetone stable for 10 days Some significant & erratic between-eye differences noted particularly with Potassium & Osmolalities

Study Cases
Deaths from Alcoholic Ketoacidosis Hyper & Hypoglycaemic Deaths Prolonged Hypoxic periods Sudden Cardiovascular Deaths

Alcoholic Ketoacidosis
Unexpected death in chronic alcoholics Acute alcohol intoxication excluded The post-mortem, Histology, Microbiology and Toxicology show no clear cause of death Liver Glycogen concentrations are low Increased Glucagon/Insulin ratio causes extensive lipolysis and ketogenesis VH -Hydroxybutyric Acid & Acetone levels

The Biochemistry of Alcoholic Ketoacidosis

Results
63 Deaths in reported Chronic Alcoholics 47 of these Deaths with VH CDT >5 U/L 8 Deaths where Blood & VH Ethanol was <20 mg/L but where VH Acetone >350 mg/L and Blood & VH -Hydroxybutyric Acid levels are >650 mg/L but <5000 mg/L. Peripheral Blood to VH Ratio -Hyd:Ace >5 will increase the number of confirmed cases to 10

Hyper- & Hypoglycaemia

Reliable measurement of post-mortem Blood Glucose is near impossible AT ANY STAGE. Stability experiments show that the detection of ANY PEG extracted Insulin in Blood or Vitreous Humour post-mortem suggests high and possibly fatal Insulin levels at the time of death. Combined VH Glucose & Lactate Concentrations more useful for Hyperglycaemic states. HbA1c reported stable post-mortem.

Results
PEG Extracted Insulin has been detected in both Blood & VH post mortem in 7 cases. Combined VH Glucose & Lactate in all the above cases <1400 mg/L suggesting Hypoglycaemia 5 cases where VH Glucose & Lactate has been >5000 mg/L in previously unknown diabetics Hb A1c was >7.5 % in 4 of the above cases

Hypoxanthine Estimation
Hypoxanthine is formed from the hypoxic degradation of adenosine monophosphate Concentrations of Hypoxanthine and cAMP in Vitreous Humour reported to give information about presence of ante mortem hypoxia Long or repeated intermittent periods of Hypoxia reported to produce VH Hypoxanthine levels some 20-50 times greater than controls

Results
In 25 unspecified accidents without any known hypoxia before death VH Hypoxanthine concentrations were always <0.1 mol/L 3 fire deaths where VH Hypoxanthine concentrations were 38-54 mol/L Confirmed Cardiac deaths have shown VH levels between 23-101 mol/L 4 VH SIDS have shown levels >300 mol/L

Cardiovascular System Respiratory System Neurological System

Unexpected & Sudden Death from Natural Causes

Intracranial Vascular Lesions

Gastrointestinal System Gynaecological Conditions

Complications of Pregnancy

Deaths from Asthma & Epilepsy

Disease processes that could lead to Cardiac Troponin T (cTnT) release

REMEMBER cTnT assay is NOT Ischaemia Specific. Does a marginally elevated cTnT reflect reversible or irreversible Cardiac damage in a patient? Cardiomyocyte necrosis, Congestive Heart Failure Pulmonary Embolism, Septic Shock, Haematoma Tachycardia, Surgery & Myocarditis Renal Failure (..sub clinical cardiac disease?) Cardiotoxicity from Cancer Therapy

Results
659 paired post mortem Blood specimens taken from 2 distinct peripheral sites for acceptance Unselected cases always used for analysis 1:3 PBSaline dilution of post mortem Blood used 87 paired specimens show cTnT >0.60 g/L The Corresponding Cardiac Pathology and/or Histopathology was seen in 58 Cases (67%) Now Best Practice guidelines from RCPath

Other Useful Biochemistry?

Cholesterol & Lipoprotein Electrophoresis Serum Cortisol, Growth Hormone TSH, Total T4 & Total T3 (..not FREE T4) Urine Catecholamines and precursors VH Amylase for Hypothermia (elevated??) CK Isoenzymes & discriminant function

Summary(1)
Post-mortem biochemical analysis should NEVER be performed as an afterthought Useful in selected cases ONLY VH Only real choice with embalmed bodies Human Tissue Act & Coroners Rules will largely prevent any research on further VH analysis VH Probably NEVER be accepted as a Forensic technique for unnatural death investigation

Summary(2)
Just when a scientific principle or discovery crosses the line between the experimental and demonstrable stages is difficult to define. Somewhere in this twilight zone the evidential force of the principle must be recognized, and while Courts will go a long way in admitting expert testimony deduced from a well-recognized scientific principle or discovery, the thing from which the deduction is made must be sufficiently established to have gained general acceptance in the particular field in which it belongs.