An Introduction to Forensic Toxicology

Transcription

1 An Introduction to Forensic Toxicology Jennifer Shea, PhD, FCACB Ken Obenson, MBBS MFFLM (UK) FRCPC (Anat) FRCPC (Forensic Path) Dip American Brd of Path (Anat & Clin/Cytopath/Forensic Path) April 11, 2015

2 What is forensic toxicology? Sample collection What testing is done? Volatile Screen Broad Spectrum Drug Screen Targeted Drug Testing Biochemical Analysis Interpreting Results General Considerations Outline

3 Toxicology is the study of the adverse effects of drugs and chemicals on biological systems Forensic toxicology is the application of toxicology for the purposes of the law Four disciplines: 1. Postmortem Toxicology 2. Human Performance Toxicology 3. Doping Control 4. Workplace Drug Testing What is Forensic Toxicology?

4 Work with coroners and pathologists to help establish the role of alcohol, drugs, and poisons in the cause of death Identify and quantify the presence of drugs and chemicals in blood and other specimens Provide advice regarding result interpretation Drug kinetics and interactions Metabolism Drug tolerance Postmortem artifacts Drug stability Postmortem Toxicology

5 For every case, pathologists should collect: Preserved blood Vitreous humor Urine (if available) Other tissues are sometimes useful depending on the nature of the case and what specimens from the list above are available: Liver Bile Stomach Contents Sample Collection: The Basics

6 Whole Blood Two FULL preserved (grey top) vacutainers (minimum 10 ml required) Preservative (NaF) prevents bacterial activity that may cause postmortem alcohol formation Preservative prevents degradation of drugs (eg cocaine) by enzymes in blood Vacutainers should be full to prevent loss of volatile substances (eg. alcohol) to air space above sample Blood obtained from the femoral vein is preferred due to the potential for postmortem drug redistribution if collected from other sites Blood should be kept at 4 C (up to one week) until sent to the toxicology laboratory Sample Collection: The Basics

7 Postmortem Drug Redistribution (PMR) Property of chemical make up of drug, volume of distribution, etc Some drugs are sequestered in tissues; very little freely circulates in blood After death, significant amounts of drug can be released from tissues (eg heart, liver, lungs) which falsely elevates blood concentration in vessels associated with these organs Tricyclic antidepressants are the classic example of drugs undergoing PMR however many reports of other drugs as well PMR may be related to the postmortem interval as well Sample Collection: The Basics

8 Postmortem Drug Redistribution: Case Reports Anatomic Location Femoral /Subclavian Time of Collection At scene Autopsy* Blood Fentanyl Concentration Case 1: 19 ng/ml Case 2: 18 ng/ml Case 1: 15 ng/ml Case 2: 39 ng/ml Heart Autopsy* Case 1: 22 ng/ml (Ratio = 1.5) Case 2: 46 ng/ml (Ratio = 1.2) Adapted from J Forensic Sci. 2014;59(5): *Autopsy occurred on average 18.8 hours after blood draw at scene. Sample Collection: The Basics

9 Urine Two preservative free (red top) vacutainers. Preserved vacutainers also acceptable. Urine may be used as a screening tool when required. Drugs/metabolites typically present in higher concentrations and remain for days after use Vitreous Humour Preservative free vacutainer. Preserved vacutainer also acceptable except when requesting electrolytes Sodium in preservative interferes with Na result Sample Collection: The Basics

10 Liver Tissue Preservative free plastic specimen container. Minimum 10 grams required. Most drugs metabolized in liver. May be useful when blood unavailable. Bile Fluid Preservative free vacutainer or plastic specimen container. Gastric contents Preservative free vacutainer or plastic specimen container. Note: Minimal data available to interpret drug concentrations in these specimen types (esp. stomach contents and bile fluid). Consult with toxicologist if testing is required. Sample Collection: Additional Specimens

11 In general, perform three different screens: 1. Alcohol analysis 2. Broad spectrum drug screen 3. Biochemical analyses (aids in identification of diabetes, kidney disease, diabetic/alcoholic ketoacidosis, etc) *Targeted drug testing available upon request (for most drugs) What type of testing is done?

12 Specimen Tests Routinely Performed Volatile screen (ethanol, methanol, isopropanol, acetone) BLOOD HbA1c (marker of diabetes) Broad Spectrum Drug Screen (includes amphetamines, anticonvulsants, antidepressants, antihistamines, antipsychotics, barbiturates, benzodiazepines, cannabinoids, CNS stimulants, cocaine and metabolites, hallucinogens, hypnosedatives, hypoglycemics, muscle relaxants, NSAIDs, opiates/opiods, salicylates) What type of testing is done?

13 Specimen VITREOUS HUMOR Tests Routinely Performed Volatile screen (ethanol, methanol, isopropanol, acetone) Glucose (marker of diabetes) Beta-hydroxybutyrate (marker of ketoacidosis) URINE OTHER (liver, bile, stomach contents, etc) Urea and creatinine (marker of kidney function) Used for screening purposes when required. Screen includes the same list of drugs as blood. Not routinely tested unless required for interpretation. When testing of a specific drug or group of drugs in these specimens is required, please contact the toxicologist to discuss. What type of testing is done?

14 Screens and (if present) quantitates four volatile substances using head space GC-FID analysis 1. Ethanol One of the most commonly abused drugs 2. Methanol Common solvent used in industrial and clinical labs 3. Acetone Ketone body produced during carbohydrate deprivation (eg. alcoholic/diabetic ketoacidosis) 4. Isopropanol Rubbing alcohol. May also be produced by acetone metabolism. Toxicology Testing: Volatile Screen

15 Death due to ethanol ingestion may be the result of: Acute ingestion Alcoholic ketoacidosis Ethanol in combination with other drugs Consumption of large amounts of ethanol results in respiratory and CNS depression in a dose-dependent manner < 50 mg/dl generally considered insignificant > mg/dl may result in coma >350 mg/dl possible death from respiratory arrest These thresholds are general guidelines. Limits may be lowered when other drugs are present. Toxicology Testing: Ethanol

16 Caveats associated with interpreting ethanol concentrations: 1. Postmortem Formation Up to 100 mg/dl ethanol (and even higher in some case reports) can be formed in blood due to fermentation of substrates (eg. glucose) in the presence of bacteria (eg. decomposed body). Ethanol also measured in vitreous humor or urine; protected fluid therefore more resistant to postmortem ethanol formation If ethanol present in blood but not vitreous, suspect postmortem formation 2. Tolerance Dispositional/Metabolic Altered absorption, distribution or elimination due to repeated ethanol use Cellular Altered pharmacological response to ethanol Toxicology Testing: Ethanol

17 Screen performed on all blood samples received using immunoassay and mass spectrometry (LC/TOF-MS) All positive results are confirmed and quantitated using a variety of analytical techniques (GC-MS, GC-MS/MS, LC-MS/MS) > 300 drugs included in screen If you want to know if a particular drug is included, call the toxicologist. Commonly encountered drugs include opiates/opioids (codeine, morphine, methadone, fentanyl), antidepessants, stimulants (cocaine, amphetamine/methamphetamine), other therapeutic medications Toxicology Testing: Broad Spectrum Drug Screen

18 Next to alcohol, most common postmortem tox finding Opiate = Naturally occuring alkaloid analgesic (eg. morphine and codeine) Opioid = Opiates + synthetic and semi-synthetic alkaloids whose pharmacological effects mimic morphine (eg. heroin, fentanyl, methadone, etc) Morphine Codeine Heroin Fentanyl Methadone Toxicology Testing: Opiates/Opioids

19 Opiates/opioids elicit their effects by binding to opioid receptors within the central and peripheral nervous system as well as GI tract Therapeutic Effects: Mainly used for pain relief (acute and chronic) Side Effects: CNS and respiratory depression, sedation, euphoria, constipation, nausea/vomiting, euphoria Additive effects with alcohol on CNS and respiratory depression Codeine < Morphine/Methadone < Heroin << Fentanyl Toxicology Testing: Opiates/Opioids

20 Tolerance is very important to consider when interpreting opioid results Best demonstrated by looking at therapeutic, toxic, and fatal ranges for methadone Therapeutic (ng/ml) In 16 drug related fatalities, mean postmortem methadone concentration was 700 ng/ml (range 110 1,310 ng/ml) In 35 non-drug related deaths, the mean postmortem methadone concentration was 640 ng/ml (range 100 4,260 ng/ml) It is almost impossible to interpret methadone results without usage history Toxicology Testing: Opiates/Opioids Toxic (ng/ml) Fatal (ng/ml) Approx Approx Approx. >300

21 Benzodiazepines (eg. diazepam, clonazepam, etc) One of the most widely prescribed drugs CNS depressants anxiolytics, anticonvulsants, muscle relaxants Rarely result in fatality unless combined with other drugs Cannabinoids Delta-9 THC is primary active metabolite Metabolites may be present in urine for many days (up to 20 days in chronic smokers) Diazepam Delta-9 THC Toxicology Testing: Other common drugs

22 Stimulants (eg. cocaine, amphetamine, methamphetamine) Not as commonly observed in postmortem cases in NB Concomitant use of alcohol and cocaine can result in production of a toxic metabolite, cocaethylene Antidepressants (eg. amitriptyline, sertraline, venlafaxine, citalopram, etc) Very common to detect antidepressant(s) on postmortem toxicology, either as incidental finding or related to an overdose Most subject to PMR interpret with caution if central blood used for analysis Cocaine Amitriptyline Toxicology Testing: Other common drugs

23 No single analytical technique can detect all drugs Not all drugs are part of broad spectrum drug screen Targeted drug testing can be done for some newer drugs (eg. bath salts, synthetic cannabinoids) or those less frequently encountered (eg. poisons such as CO) Important to provide case history with all toxicology requests! Methodology used depends largely on the drug s chemical structure. GC-MS, GC-MS/MS, LC-MS/MS, etc Toxicology Testing: Targeted Drug Testing

24 Laboratory also performs routine biochemical analyses on specimens as an adjunct to toxicology testing Vitreous humor is best specimen for most biochemical testing as blood is usually hemolysed which may interfere with analytical methodologies Exception: HbA1c in whole blood Biochemical testing aids in identification of undiagnosed or poorly controlled diabetes (VH glucose, WB HbA1c), diabetic ketoacidosis (VH β-oh, glucose) and kidney function (VH creatinine, urea) Toxicology Testing: Biochemical Analyses

25 Diabetes is a disease characterized by hyperglycemia Complications are classified as chronic (nephropathy, retinopathy, neuropathy) and acute (diabetic ketoacidosis) Estimated that 1 million have the disease but do not know it Biochemical Analyses: Diabetes Public Health Agency of Canada, 2011 Canadian Diabetes Association

26 Insufficient cellular glucose to maintain energy stores Adipose tissue breaks down fatty acids Liver converts fatty acids to ketone bodies as alternative energy source: Beta-hydroxybutyrate Acetoacetate Acetone Liver also breaks down glycogen into glucose END RESULT: DKA GLUCOSE and KETONE BODIES ph (metabolic acidosis)

27 Extremely difficult to interpret post-mortem blood glucose Continuous consumption of glucose by surviving cells and bacteria post-mortem Glucose almost always undetectable in blood Blood glucose rarely measured in forensic chemistry Vitreous humor is better preserved after death making it a better specimen choice High VH glucose suggestive of high plasma glucose at time of death Caveats with VH glucose testing: Ongoing metabolism occurs in VH too so will also see drop postmortem Low VH glucose Hypoglycemia Biochemical Analyses: Blood vs Vitreous Humor Glucose

28 β-hydroxybutyrate (βhb) Ketone body produced during ketoacidosis Measured in vitreous humor Can help elucidate chronic hyperglycemia from acute diabetic ketoacidosis (>3 mmol/l generally indicative of ketoacidosis) HbA1c Formed through non-enzymatic addition of glucose to N-terminal valine of β chain of Hb Marker of longer term (~3 mth) glycemic control Reflects metabolic state of the deceased in the weeks prior to death More stable than glucose in blood (internal studies show stability up to 6 mth) Can support other findings including elevated glucose and βhb HbA1c 6.5% consistent with diabetes Biochemical Analyses: Other Markers of Diabetes

29 Evidence of drug overdose or non-compliance with prescribed medication? State of body on discovery Decomposition may result in decreased (stability) or increased (PMR) drug levels Postmortem formation of ethanol Source of blood (central vs peripheral)? Tolerance to drugs detected in blood? Opiates/opioids Ethanol Interpreting Results: General Considerations

30 Forensic toxicology can aid pathologists and coroners in elucidating the cause and manner of death Proper specimen collection is vital to obtaining meaningful toxicology results Preserved vacutainers help maintain specimen integrity of blood samples Peripheral (femoral) blood is always preferred over a central source A case history (including medication summary) and/or preliminary autopsy findings can greatly aid the toxicologist Can aid with interpretation of drug levels where tolerance is important Can help determine need for additional testing above and beyond general drug screen When in doubt, call the toxicologist! Summary

31 Please contact me with any questions. Phone: Thank you!