Postmortem Forensic Toxicology

1
Postmortem Forensic Toxicology

• Teri Martin
• teri.martin_at_jus.gov.on.ca
• September 23, 2003

2
Outline

• Definitions and purpose of postmortem tox
• Samples of forensic interest
• Handling and storage of samples
• Pitfalls in postmortem toxicology
• Interpretation of results

3
Postmortem Forensic Toxicology

• Qualitative and quantitative analysis of drugs or
  poisons in biological specimens collected at
  autopsy
• Interpretation of findings in terms of
• Physiological effect at time of death
• Behavioural effect at time of death

4
Quantitative vs. Qualitative

• Qualitative analysis determines the presence or
  absence of a drug or poison in a submitted sample
• Quantitative analysis determines the amount of
  drug or poison that is present in the submitted
  sample

5
Postmortem Forensic Toxicology

• Types of cases
• Suspected drug intoxication cases
• Fire deaths
• Homicides
• Driver and pilot fatalities
• Therapeutic drug monitoring
• Sudden infant death (SIDS)

6
Samples of Forensic Interest
7
Issues in Specimen Collection

• Selection
• Multiple, varied sites of collection
• Collection
• Appropriate method of collection
• Adequate volumes for analysis
• Storage and handling
• Important to ensure analytical results are
  accurate and interpretations are sound

8
Typical autopsy specimens

• Blood
• Urine
• Stomach contents
• Bile
• Liver
• Hair
• Vitreous humor

9
Blood

• Antemortem ? ideal blood sample
• Postmortem blood is not truly blood
• Anatomical site of collection at autopsy should
  be noted

10

• Central sites
• Heart
• Peripheral sites
• Femoral
• Iliac
• Subclavian
• Other sites
• Head blood
• Hematoma blood

Subclavian
Heart
Iliac
Femoral
11
Hematoma

• Extravascular blood clot
• Protected from metabolism
• Analysis will indicate what drugs were present in
  the blood at the time of formation

12
Hematoma case example

• A 26 year old man was found dead at the bottom of
  a staircase. Death was due to physical injuries.
• Question as to alcohol use prior to fall down
  stairs
• No urine available at autopsy
• Alcohol not detected in femoral blood
• Alcohol in hematoma blood ? 150 mg/100 mL
• The deceased had been drinking prior to receiving
  the head trauma.
• The deceased had survived for several hours after
  the injury.

13
Hematoma

• Caution There may be a delay between the
  incident which resulted in hematoma and the
  actual formation of the hematoma
• Therefore, this alcohol concentration does not
  necessarily indicate the BAC at the time of the
  fall down the stairs.

14
Urine

• Produced by the kidneys
• Blood filtered by the kidneys
• Stored in the bladder until voided
• Qualitative - the presence of a drug in the urine
  of an individual indicates that some time prior
  to death the drug or poison was present in the
  blood of the individual

15
Stomach contents

• Visual examination may reveal tablets
• Drugs that have been orally ingested may be
  detected in stomach contents
• Caution drugs administered by other routes may
  also diffuse into stomach contents from the blood
• Generally qualitative
• Stomach contents are not homogeneous
• Only a portion of stomach contents collected
  (unmixed?)
• Useful for directing further analysis

16
Case Example

• A 26 year old woman is found dead in bed
• Numerous medications in her home
• Amitriptyline, Oxycodone, Morphine, Paroxetine,
  Diphenhydramine, Pseudoephedrine, Phenobarbital,
  Codeine, Temazepam, Diazepam
• Only 3 mL of blood collected at autopsy
• Qualitative analysis of stomach contents
• Amitriptyline detected
• Nortriptyline detected
• Quantitation can now be performed in blood

17
Liver

• Drug metabolism occurs in the liver
• Both parent compounds and metabolites may be
  present in higher concentrations in the liver
  than in the blood ? ease of detection
• Limitation is that drugs are not uniformly
  distributed throughout the liver ? confounds
  interpretation

18
Bile

• Digestive secretion
• Continuously produced by the liver
• Stored in the gallbladder
• Qualitative - the presence of a drug in the bile
  of an individual indicates that sometime prior to
  death, the individual was exposed to the drug

19
Vitreous humor

• Fluid that occupies the space between the lens
  and the retina of the eye.
• Sequestered from putrefaction, charring and
  trauma, microorganisms.
• Useful in cases where decomposition is advanced,
  body is exhumed or in fire deaths
• Limitation is bloodvitreous ratio may not be
  known

20
Hair

• Recent specimen of interest
• Metabolism does not occur in hair
• Can provide a historical record of drug or poison
  exposure
• Pros and cons of hair analysis still being
  uncovered ? racial variability?

21
Case Example
Poklis, A. 2002. Abstract SOFT, Dearborn,
Michigan.

• 30 year old woman, previously in good health
• Nausea, vomiting, diarrhea, rash, fever
• Weakness in hands and feet ? Guillian Barre?
• Hospitalized with hypotension, seizures
• Misplaced laboratory result ? Arsenic!
• Sequential hair analysis for arsenic showed
  chronic arsenic poisoning over 8 month period

22
Non-biological submissions

• Used to direct analysis of biologicals
• May indicate the nature of substances that may
  have been ingested, inhaled or injected
• Examples
• Containers found at the scene
• Syringes
• Unidentified tablets or liquids

23
Autopsy specimens of limited value

• Pleural fluid
• Chest cavity blood
• Gutter blood
• Samples taken after embalming
• Samples taken after transfusion in hospital

• Spleen squeezings
• Esophageal scrapings

24
Chest Cavity Fluid

• Not readily definable
• Most likely to be collected if
• Traumatic injury to the chest
• Advanced decomposition
• A contaminated blood sample, chest cavity fluid
  may contain fluids from stomach, heart, lungs etc.

25
Samples taken after embalming

• Methanol is a typical component of embalming
  fluid
• Most drugs are soluble in methanol
• Embalming process will essentially wash the
  vasculature and tissues
• Qualitative analysis can be performed on body
  tissues

26
Case Example

• A 72 year old woman, given meperidine to control
  pain following surgery, later died in hospital.
  The woman was in poor health and it is possible
  that death was due to natural causes. However,
  coroner requests toxicology to rule out
  inappropriate meperidine levels.
• BUT
• Body had been embalmed
• Liver and spleen submitted

27
Storage and Handling
28
Proper specimen handling

• Identification of samples
• Continuity
• Contents
• Specimens delivered to lab without delay
• Specimens should be analyzed as soon as possible
• Storage areas should be secure

29
Storage and Handling

• Not feasible to analyze specimens immediately
• Sample should be in well-sealed container
• Sample containers must be sterile
• Use of preservatives and anti-coagulants
• Refrigeration vs. Freezing
• Both inhibit bacterial action esp. freezing
• Freezing results in ? prep time
• Freeze-thaw cycle may promote breakdown

30
Storage of Samples

• Preservative
• Sodium fluoride
• Anti-coagulants
• Sodium citrate
• Potassium oxalate
• EDTA
• Heparin
• Not imperative for postmortem blood samples

31
Determining analyses

• Case history
• Medical history
• Autopsy findings
• Symptomatology
• Experience of the toxicologist
• Amount of specimen available
• Nature of specimens available
• Policies of the organization

32
Pitfalls in Postmortem Forensic Toxicology
33
Decomposition

• Autolysis
• The breakdown of cellular material by enzymes
• Putrefaction
• A septic/infectious process
• The destruction of soft tissues by the action of
  bacteria and enzymes
• Traumatic deaths may demonstrate ? putrefaction

34
Decomposition

• Fewer samples available for collection
• Quality of samples is diminished
• Putrefaction produces alcohols
• Ethanol
• Isopropanol
• Acetaldehyde
• n-propanol

35
Postmortem redistribution

• A phenomenon whereby increased concentrations of
  some drugs are observed in postmortem samples
  and/or site dependent differences in drug
  concentrations may be observed
• Typically central blood samples are more prone to
  postmortem changes (will have greater drug
  concentrations than peripheral blood samples)

36
Possible mechanisms of postmortem redistribution

• Diffusion from specific tissue sites of higher
  concentration (e.g. liver, myocardium, lung) to
  central vessels in close proximity
• Diffusion of unabsorbed drug in the stomach to
  the heart and inferior vena cava
• Diffusion of drugs from the trachea, associated
  with agonal aspiration of vomitus

37
Case Example

• 37 year old man found dead in his home
• Cause of death identified at autopsy as asphyxia
  due to choking white pasty material lodged in
  throat

• Heart blood
• Morphine 20 000 ng/mL
• Amitriptyline 0.36 mg/dL

• Femoral blood
• Morphine 442 ng/mL
• Amitriptyline 0.01 mg/dL

• Examination of esophageal and tracheal contents
  revealed presence of both morphine and
  amitriptyline

38
Susceptible Drugs

• Drugs most commonly associated with postmortem
  redistribution
• are chemically basic
• have large volumes of distribution

39
Volume of distribution

• Review from last lecture
• Volume of distribution is the amount of drug in
  the whole body (compared to the amount of drug in
  the blood)
• If a drug has a large volume of distribution, it
  is stored in other fluids and tissues in the body

40
Susceptible Drugs

• Tricyclic antidepressants
• Amitriptyline
• Nortriptyline
• Imipramine
• Desipramine
• Antihistamines
• Diphenhydramine

• Narcotic Analgesics
• Codeine
• Oxycodone
• Propoxyphene
• Doxepin
• Digoxin

41
Example Digoxin
p. 60, Principles of Forensic Toxicology

• A 33 year old white female is admitted to
  hospital after taking 60 digoxin tablets
• An antemortem blood sample collected 1 hour prior
  to her death indicates a blood digoxin level of
  18 ng/mL
• Heart blood digoxin concentration obtained at
  autopsy is 36 ng/mL

42
Example Digoxin

• Postmortem increase in blood digoxin
  concentrations is suspected to be due to the
  release of the drug from the myocardium
• Postmortem levels gt Antemortem levels
• Heart blood levels gt Femoral blood levels

43
Postmortem redistribution

• Coping with the problem of postmortem
  redistribution
• Analysis of both central blood and peripheral
  blood in cases where postmortem redistribution
  may be a factor
• Compilation of tables to determine average and
  range of postmortem redistribution factors for
  drugs

44
Incomplete Distribution

• Site dependent differences in drug levels due to
  differential distribution of drugs at death
• Has been noted in rapid iv drug deaths
• Example
• Intravenous injection of morphine between the
  toes
• Fatal amount of drug reaches the brain
• Full distribution of the morphine throughout the
  body has not occurred
• Femoral concentration gt Heart concentration

45
Drug Stability

• Knowledge of a drugs stability is necessary to
  facilitate interpretation of concentrations
• Breakdown of drugs may occur after death and
  during storage via non-enzymatic mechanisms
• Cocaine ? Benzoylecgonine (Hydrolysis)
• LSD ? degradation due to light sensitivity
• Others ?

46
Example Bupropion

• Bupropion, an antidepressant, was identified and
  confirmed during a GC drug screen
• Blood analyzed using a quantitative analysis
• Bupropion ? not detected
• Review of the literature
• Laizure and DeVane, 1985. Ther. Drug. Monit.
• Bupropion showed a log linear degradation that
  was both temperature and pH dependent

47
Evaporation of volatiles

• Ethanol
• Carbon monoxide
• Cyanide
• Toluene
• Other alcohols

48
Example Carbon Monoxide
Ocak et al. 1985. J. Analytical Toxicology. 9
202-206

• Effects of storage conditions on stability of CO
• No significant change in CO saturation in
  capped samples stored at room temperature or 4oC
• Significant losses in CO saturation in uncapped
  samples stored at room temperature and at 4oC
• Mechanism for loss ? diffusion

49
Interpretation
50
Interpretation

• Therapeutic, toxic or fatal? How do you know?
• Compare measured blood concentrations with
  concentrations reported in the literature
• Clinical pharmacology studies
• Incidental drug findings
• Plasma ? blood
• Consider case history
• Symptoms observed by witnesses?
• Tolerance of the individual to the drug

51
Bloodplasma ratios

• Knowledge of the bloodplasma ratio can be very
  important when applying information from clinical
  studies to postmortem forensic tox
• Cocaine, bloodplasma ratio is 1.0
• Phenytoin, bloodplasma ratio is 0.4
• Ketamine, bloodplasma ratio is 1.7
• Hydroxychloroquine, bloodplasma ratio is 7.2

52
Example THC

• Six healthy male volunteers recruited for a study
  of the pharmacokinetics of THC in humans
• Smoked a high-dose THC cigarette
• 15 minutes after cessation of smoking, plasma THC
  concentrations averaged 94.8 ng/mL
• The plasmablood ratio for THC is 1.8
• Plasma contains 1.8x as much THC as whole blood
• The results of this study correspond to a blood
  THC concentration averaging 53 ng/mL

53
Importance of History Tolerance

• Drug concentrations in non-drug related deaths
  may overlap with reported drug concentrations in
  fatal drug intoxications
• Methadone example
• Naïve users - deaths due to methadone are
  associated with blood levels gt 0.02 mg/100 mL
• Patients on methadone maintenance peak blood
  concentrations may range up to 0.09 mg/100 mL

54
Interpretation

• Acute vs. Chronic Ingestion Can you tell?
• Parentmetabolite drug concentration ratio may
  be of assistance in differentiating between acute
  and chronic ingestion of a drug

55
Example Amitriptyline

• Case 1
• Amitriptyline 0.4 mg
• Nortriptyline 0.02 mg
• Parent gtgt Metabolite
• Suggestive of acute overdose and rapid death

• Case 2
• Amitriptyline 0.04 mg
• Nortriptyline 0.08 mg
• Parent lt Metabolite
• Slow death and/or chronic administration

56
Interpretation

• Metabolites are produced when drugs are
  biotransformed (converted) into other chemicals,
  more easily excreted from the body
• Metabolite drug concentrations may be the more
  useful measure of exposure or toxicity

57
Metabolites Exposure

• The parent compound may be a prodrug or may have
  a shorter t1/2 than the metabolite
• Clorazepate ? nordiazepam
• Flurazepam ? N-desalkylflurazepam
• Heroin ? morphine

58
Metabolites Toxicity

• The metabolite may have ? toxicity over the
  parent compound
• Acetaminophen ? N-Acetylbenzoquinoneimine
• Meperidine ? normeperidine
• Methanol ? formic acid
• Ethylene glycol ? oxalic acid ? calcium oxalate