Postmortem Forensic Toxicology by gjjur4356

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									         Postmortem
         Forensic
         Toxicology

        Teri Martin
teri.martin@jus.gov.on.ca
    September 23, 2003
                   Outline

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

Types of cases:
• Suspected drug intoxication cases
• Fire deaths
• Homicides
• Driver and pilot fatalities
• Therapeutic drug monitoring
• Sudden infant death (SIDS)
Samples of Forensic Interest
         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
       Typical autopsy specimens

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

• Antemortem  ideal blood sample

• Postmortem blood is not truly “blood”

• Anatomical site of collection at autopsy
  should be noted
                           • Central sites
              Subclavian
                             • Heart

          Heart
                           • Peripheral sites
                             • Femoral
 Iliac
                             • Iliac
                             • Subclavian
Femoral

                           • Other sites
                             • Head blood
                             • Hematoma blood
                   Hematoma


• Extravascular blood clot
• Protected from metabolism
• Analysis will indicate what drugs were
  present in the blood at the time of formation
            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.
                   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.
                    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
                 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
                   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
                    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
                    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
            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 blood:vitreous ratio may not
  be known
                    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?
                   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
      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
      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”
                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.
      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
                  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
Storage and Handling
         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
           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
            Storage of Samples

• Preservative
  • Sodium fluoride
• Anti-coagulants
  •   Sodium citrate
  •   Potassium oxalate
  •   EDTA
  •   Heparin
  •   Not imperative for postmortem blood samples
            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
Pitfalls in Postmortem
 Forensic Toxicology
                 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
                Decomposition

• Fewer samples available for collection
• Quality of samples is diminished
• Putrefaction produces alcohols
  •   Ethanol
  •   Isopropanol
  •   Acetaldehyde
  •   n-propanol
         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)
            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
                     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                   • Femoral blood
  • Morphine: 20 000 ng/mL        • Morphine: 442 ng/mL
  • Amitriptyline: 0.36 mg/dL     • Amitriptyline: 0.01 mg/dL

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

Drugs most commonly associated with postmortem
redistribution:

   1. are chemically basic
   2. have large volumes of distribution
            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
                Susceptible Drugs

• Tricyclic             • Narcotic
  antidepressants         Analgesics
  •   Amitriptyline       • Codeine
  •   Nortriptyline       • Oxycodone
  •   Imipramine          • Propoxyphene
  •   Desipramine

• Antihistamines        • Doxepin
  • Diphenhydramine     • Digoxin
                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
               Example: Digoxin


• Postmortem increase in blood digoxin
  concentrations is suspected to be due to the
  release of the drug from the myocardium

  • Postmortem levels > Antemortem levels
  • Heart blood levels > Femoral blood levels
         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
           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 > Heart concentration
                   Drug Stability


• Knowledge of a drug’s 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 ?
              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…”
          Evaporation of volatiles


•   Ethanol
•   Carbon monoxide
•   Cyanide
•   Toluene
•   Other alcohols
         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
Interpretation
                       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
                 Blood:plasma ratios

• Knowledge of the blood:plasma ratio can be
  very important when applying information from
  clinical studies to postmortem forensic tox

  •   Cocaine, blood:plasma ratio is 1.0
  •   Phenytoin, blood:plasma ratio is 0.4
  •   Ketamine, blood:plasma ratio is 1.7
  •   Hydroxychloroquine, blood:plasma ratio is 7.2
                   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 plasma:blood 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
       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 > 0.02 mg/100 mL
  • Patients on methadone maintenance – peak blood
    concentrations may range up to 0.09 mg/100 mL
                 Interpretation


Acute vs. Chronic Ingestion: Can you tell?

• Parent:metabolite drug concentration ratio may
  be of assistance in differentiating between
  acute and chronic ingestion of a drug
           Example: Amitriptyline

         Case 1                    Case 2

Amitriptyline: 0.4 mg%     Amitriptyline: 0.04 mg%
Nortriptyline: 0.02 mg%    Nortriptyline: 0.08 mg%

  Parent >> Metabolite       Parent < Metabolite

Suggestive of acute        Slow death and/or
overdose and rapid death   chronic administration
                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
          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
             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

								
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