DR A J JEFFERY

MBChB MD FRCPath (Forensic) MFFLM

HOME OFFICE REGISTERED FORENSIC PATHOLOGISTPost-mortem Toxicology

Areas to CoverWhy take toxicologyWhat samplesHow to take themWhat does the toxicologist do with the samples?How to interpret the results general considerationsAlcoholDrugs of abuseToxicology in other causes of deathOther specimensCase examples

WHY TAKE TOXICOLOGY?

Why take toxicology ?To ascertain if the deceased was under the influence of alcohol or drugs of abuse at the time of their death.RTAs / Accidental deaths / suicides

To confirm or refute overdose / poisoning

To confirm presence / levels of therapeutic drugs.Eg epilepsy / antidepressants

WHAT SAMPLES ARE APPROPRIATE ?

SamplesBlood (plain)(peripheral)Blood(preserved)(peripheral)Urine(plain)Urine(preserved)

Fluoride inhibits further alcohol production but wont undo the damage already done.

VitreousStomach Contents

TissuesLiver (mid R lobe)Skeletal muscle (eg psoas) (if embalmed buttock)

OBTAINING THE BLOOD SAMPLE

Femoral Vein SamplingVein NOT arteryBefore eviscerationBefore urine sampling

Ideal = tie off / clamp, then sample by wide-bore needle belowRoutine = clean catch

Ideal = dont milk the legRoutine = required to gain sufficient sample

MINIMAL FEMORAL BLOODProblem:

Insufficient Femoral BloodTake what ever you can in preserved tubesSubclavian = reasonable alternative

Could take free-lying chest blood etc for screening for the general presence of drugs

Make sure you say where each sample has come from

Obtain an alternative specimen

OBTAININGTHE URINE SAMPLE

Urine SamplingNeedle and syringe orOpen dome of bladder and aspirate with syringe alone

Presence of a catheter may be important toxicologically as the urine may contain artefactually high lignocaine due to catheter lubricant gel

Vitreous

WHAT DO THE TOXICOLOGISTS DO WITH THE SAMPLE?

AnalysisScreening (GC / Immunoassay)What classes of drug are present

Confirmation (GCMS)Specific drugs found by breaking them down & looking at the breakdown products.

QuantificationTechnique may vary dependent on the nature of the drug being analysed.

HOW TO INTERPRET THE RESULTS

General ConsiderationsAccuracy of reference rangesRe-distribution site matters!Individual variation (e.g. renal disease)DecompositionTolerance

Accuracy of reference rangesInterpretation of absolute drug levels / Reference ranges

Based on individual reports

Variable from lab to lab due to varying techniques

Need to consider the previous list

General ConsiderationsRe-distribution site matters!Individual variation (renal disease)DecompositionTolerance

RedistributionDiscovered with digoxinMost drugs that undergo redistribution do so because of their relative lipid solubility.Due toLoss of cell integrityDiffusionGI tract to adjacent structuresThrough conduits lymphDiffusion from bladder

Natural DiseaseDepends or route of administration1st pass / second pass metabolism

AbsorptionWith or without mealGI surgery

Elimination / ClearanceRenal impairmentLiver impairment

DecompositionSignificant redistribution

Some drug levels increaseAlcohol production by bacterial action

Others degrade

If there is a degree of decomposition make sure you write it on the tox request

ToleranceIncreasing doses required over time to achieve same effects.What is lethal to a nave user may have no effect at all in a chronic user.

First dose deaths

People walking around and working with enough drugs on board to kill an elephant!

Prison release deaths

Alcohol

Deaths due to AlcoholWhat alcohol related causes of death do you know?

How might you classify them?

Which specific toxicological causes do you know?

AlcoholAcute alcohol toxicity

Ketoacidosis

Alcohol in combination with other drugs

Problems with Interpretation of AlcoholRedistribution

Dealing with decomposition

Back calculations

Acute alcohol toxicityHow does it cause death?Death respiratory depression due to action on brainstem

UK legal driving limit?Driving limit 80 mg/100ml

Less than 20 mg/100ml generally considered insignificant.

>30 = higher skills30 50 = deterioration in driving50 100 = inhibitions / laughter100 150 = slurring, insteadiness, poss nausea150 200 = obvious drunkenness, nausea staggering200 300 = stupor, vomiting, coma300 + = stupor, coma, aspiration &

Alcohol fatal level or not?LD 50 = 400 mg/100ml

Alcohol has symbiotic relationship with other drugs. e.g. < 200mg/100ml can be fatal if opioids are taken.> 200mg/100ml can the fatal dose of opioids> 100mg/100ml may enhance heroin toxicity

Ethyl glucuronide (minor breakdown product) in urine if imbibed within 5 days of death.

What is ketoacidosis?Can you explain why this happens?

KetoacidosisBrain can utilise ketone bodies when glucose is unavailable fasting / starvation

Ketone bodies, formed by the breakdown of fatty acids and the de-amination of amino acids.

Ketoacidosis is an extreme and uncontrolled form of ketosis, which is a normal response to prolonged fasting. In ketoacidosis, the body fails to adequately regulate ketone production causing such a severe accumulation of keto acids that the pH of the blood is substantially decreased.

Alcoholic ketoacidosisMetabolic acidosisMalnutritionBinge drinking superimposed on chronic alcohol abuse

KetoacidosisKetones:Acetone (can be produced pm)

Alcoholic vs DiabeticHow might you differentiate?

Urine glucose

HbA1c4 - 6.1%

CalculationsAVOID !

Clearance10 25 mg/dl/hr ( about a unit an hour)In 10 hours you can clear ~ 100-200 mg/dlAlcoholics can clear 30 40 mg/dl/hr (due to training!)

Widmark equationUsed by some to predict amount of alcohol consumed

Decomposition70 190 mg/100ml reported as artefactConsider pm findingsLook for other substances produced pmUse vitreous and urine as supportive evidence

These are relatively protected from redistribution

Normal ratios (if in equilibrium)

Urine : BloodVitreous : Blood1.23 : 1 1 : 0.81

OPIOID AGONISTS

SYMPATHOMIMETICS

Drugs of Abuse

Opioid agonists

Opioid agonists

Analgesia / euphoria / dysphoria

Respiratory depression

miosis

Morphine and other opioidsMorphineHeroin (diamorphine) IV, smoked, sniffedMethadone (green liquid oral or IV)Pethidine, buprenorphine

** CNS depression **

FindingsHistory / scene / paraphernalia

External iv sitesFoam at nose / mouth

Limited & non specificPulmonary congestion and oedemaStomach contents methadone is usu green!

Morphine / HeroinHeroin / diamorphine synthetic morphine derivativePowerful opioid analgesic

Metabolised almost immediately (10 15 mins) to 6 monoacetyl morphine 6MAM and then within 24 hours to morphine.

Presence of 6MAM is consistent with use within 12-24 hoursIe recent intake / top up injections

Acute alcohol intoxication potentiates the effects

Total morphine : Free MorphineGives some idea of time since administrationEg in IV admin15 mins post admin4:160 mins9:1

TherapeuticLethal_______ Free morphine10 100ng/ml50 4000 ng/ml

HeroinA contaminate of street heroin is acetylcodeineHence may have +ve codeine levels

Most heroin deaths occur several hours after taking the drugSleepy / snoringMay have time to metabolise drug despite irreversible respiratory depression

MethadoneTherapeutic75 1100 ng/mlToxic200 2000 ng/mlLethal400 2000 ng/ml

Significant overlapTolerance becomes very importantInterpretation requires knowledge of drug historyLong & variable T

MethadoneBreakdown product EDDPThis is inactive

Titration is importantMany deaths occur during first few weeks of treatmentCan cause respiratory depression at therapeutic doses

Lipophilic so undergoes significant redistributionEven peripheral samples can be 2x in and 3x in

OpioidsTolerance = V V important considerationEg. Prison releasePalliative care

Nb worth remembering that 10% of codeine will breakdown to become morphine.

TherapeuticLethal_______ Free Codeine30 340ng/ml>1600 ng/ml

Sympathomimetics

Sympathomimetics Incr activity of adrenaline and serotonin

AdrenalinHypertensionTachycardiaMydriasisSerotoninExcitement Hyperthermia

StimulantsCocaineAmphetamineEcstasyOther methamphetamines

Associated with subarchnoid haemorrhage80% of these assoc with aneurysms

Intracerebral haemorrhageAssociated with AVMs & hypertension

FindingsHearts of stimulant users tend to be heavier than controlsFibrosisContraction band necrosisAccelerated atherosclerosisNon specific pulmonary changes

Crack cocaine smokers prominent anthracosis esp in alveolar macrophages & emphysematous changes.

CocaineNaturally occurring plant alkaloid stimulantSnorted, smoked, cutaneous, injectedNb always consider in sudden death in the same way that you might consider HOCM.

Breakdown ProductsBenzoylecgonineMethylecgonineCocaethyleneInactiveAs active as cocaine itself & indicative of alcohol consumption at the same time

Cocaine ToxicityLess than 50 ng/ml cocaine is considered not to produce measurable physiological effectsT can be as little as 40 minsBenzoylecgonine 1-4 days in urine

Toxic>900 ng/mlLethal>1000 ng/mlBenzoylecgonineLethal - >1000 ng/ml *

CocaineBut lethal nature not dose related

Long term effects:Cardiovascular damage incr ischaemic event / Coronary Art thrombosiscoronary artery spasmcontraction band necrosisfibrosis / sudden arrhytmiamyocarditis/cardiomyop/valvular/aortic dissection/hypertensive crisisNon cardiac - Cerebral infarction / intracerebral haemorrhage

So death can be attributed to cocaine even if not found in blood.

Cocaine can decrease rapidly in unpreserved blood samples stored at room temperature.

Cocaine, death & Excited deliriumExcited deliriumHyperthermiaMental & physiological arousalExcited, erratic & sometimes bizarre, violent behaviourMay have florid psychosisMay exhibit extra-ordinary strength

Tends to result in sudden respiratory arrestBlood cocaine and benzoylecgonine may be low but there is usually concentration of benzoylecgonine within the brain indicating long term useMarked decease in D2 receptors in hypothalamus in psychotic cocaine abusers.D2 receptors play a role in temperature regulation

AmphetaminePrevalence second only to cannabisSynthetic stimulantEffects similar to cocaineStimulate release of catecholamines, particularly adrenalineTolerance & dependence developAbsorbed by GIT, clinical effects commence within 20 minutes, last 4-6 hours.

AmphetamineToxic >500 ng/ml amphetamine >1800 ng/ml methamphetamineLethalUsu > 1000 ng/ml amphetamineBut can be seen if >50ong/mlUsu > 10 000 ng/ml methamphetamine

Different FormsAmphetamine (Benzedrine, uppers, 'A', speed, whizz, cranks, wake-up, sulph, hearts)

Dextroamphetamine (Dexedrine, dex, dexy, dexies)

Methamphetamine (ICE, crystal, glass, meth)

Methylenedioxyamphetamine (MDA, EVE)

3 ,4, Methylenedioxymethamphetamine (MDMA, ADAM, Ecstasy, 'E', doves, Dennis)

AmphetamineAlcohol can potentiate effects on the heart.Rare toxic effects:ComaCerebral vasculitisCerebral haemorrhageRhabdomyolysisD.I.C.Renal dysfunctionCardiac long term usersAccelerated coronary atherosclerosisMicrovascular disease

MDMA / EcstasyAmphetamine-like drugs3,4-methylenedioxymethamphetamineSerotinergic and noradrenergic effects

Hyperthermic effects

Liquid ecstasy GHBGamma hydroxybutyrate

Other

BenzodiazepinesDiazepam 20 4000 ng/mlNordiazepam 20 1800 ng/mlNeed in the order of thousands to consider fatal.

CannabisCannabinoidsTHC tetrahydrocannabinolDelta 9 THC carboxylic acid

Rapidly distributed into tissuesBlood levels drop >90% within 2 hours of intakeTHC can only be found within 4-12 hours post intake>2 ng/ml suggestive of recent intake

THC metabolites remain inBlood up to ~ 5 daysUrine up to ~ 12-36 days

Volatile substance abuse - VSAAdhesives, aerosols, petrols, paint stripper, nail varnish remover .. amongst othersIncreased risk taking behaviourAccidental suffocationCNS depressionDeaths thought to be due to cardiac arrhythmiasSensitisation of myocardium to effects of adrenalineDeaths often seen in association with physical exertionBlood sample must be in a glass tube with a foil top filled to the top.Tie off whole lung and place within a nylon bag.Head space

New DrugsMephadroneCream

IN

OTHER

CAUSES OF DEATHToxicology

Fire deathsCarboxyhaemoglobin Normal 48%

CyanideNormal < 0.1mg/LDevelops within the potted blood sample if not preserved

Carbon MonoxideIn an individual breathing airT = 4 hours

Breathing O2 in HospitalT = 60 minutes

Therefore always consider survival time.

Therapeutic DrugsAnti-depressantsAnti-convulsants

OverdoseAspirinTherapeutic20 100mg/lToxic>150 mg/lLethal>500 mg/l

NB those on reg Rx (eg arthritis 3g/day) 44-330mg/LT up to 36 hours in massive OD

FindingsPmBlood stained gastric content / frank haematemesisRarely skin petechiaeMucosal gastric erosionsMalaena if survival sufficiently longPetechiae through other organs due to anticoagulant effectesp parietal pleura and epicardium

ParacetamolTherapeutic10 20 mg/l

Toxic>150 mg/l

Lethal>160 mg/l

IMMUNOLOGY ANAPHYLAXISBIOCHEMISTRY DIABETESOther samples of interest

Immunology - AnaphylaxisSecure ante-mortem samplesNeeds to be peripheral as mast cell rich organs can release tryptase after death.

Serum (plain tube spun down)

Mast cell tryptase ( = more specific)Specific IgEMake sure you give details of any suspected causeAvailable for venoms, foods, medicines, contrast agents, latex.

Mast cell tryptaseT during life is ~ 2 hours so may be unhelpful if they have been resuscitated and have survived and die later ( usu from cerebral anoxia)Antemortem!!!

Tryptase is a sensitive marker for mast cell activationHigh levels will be found post severe anaphylaxisLevels are not raised in local allergic reaction eg rhinitisCan be raised in pure asthma deaths but not of the same order of magnitudeSlight increases can be seen in non-anaphylactic mast cell degranulation EG opioids for chest painTrauma disruption of mast cell rich tissues

Unless grossly elevated interpret with cautionIn presence of suggestive history and absence of pm findings it may provide confirmatory evidence.

Normal Levels:

IgE 0 122 kU/LMCT 2-14 mg/L

MCT can be produced pm

Biochemistry - DiabetesVitreous is best for biochem as most blood is already haemolysedGlucose drops significantly after deathBacterial metabolismDrops even in vitreousSo low glucose hypoglycaemiaIt is not possible to diagnose hypoglycaemia accurately at pm.A high vitreous glucose virtually rules out hypoglycaemia as one can assume it was the same or higher in the antemortem period unless peri-mortem dextrose admin

HbA1c heparinised sample

InsulinIf endogenous the body has to cleave C peptide from pro-insulin to make active insulin.If exogenous the insulin is already cleaved and so they will have no C peptide.

Case Examples

Case 160 yr old male, in house fire, extensive burns, no suspicious injuries, no soot in airways or stomach.RangesNormToxLethCO = 40%50%

Cyanide = 0.5 mg/L150mg/l>160mg/l6mg/l

Codeine30-340ng/ml>1600ng/ml56ng/ml

Morphine (free)10-100ng/ml50-4000ng/ml

Case 230 year old female found on floor with green fluid trailing from corner of mouth

EthanolBlood181 mg/100mlsUrine244 mg/100mlEthylglucuronide present in urine

Acetone negativeMethanolnegativeIsopropanolnegative

What effect might you expect?What caveat would you include?

Is this likely to by PM alcohol production?

Ranges:NormalToxicLethal

Methadone75-1100ng/ml200-2000ng/ml400-2000ng/ml413 ng/mlEDDP = 276 ng/ml

Amphetamine>500ng/ml>1000ng/ml471 ng/ml

Diazepam20-4000ng/ml>5000ng/ml>30 000ng/ml21 ng/mlNordiaz = 73ng/ml20-1800ng/ml

Cause of Death??

Case 3Chronic alcoholic found dead at home, head injury consistent with fall to the ground.EthanolBlood16 mg/100mlsUrine72 mg/100mlsVitreous25 mg/100mlsStom Cont145 mg/100mls

AcetoneBlood3mg/100mlsUrine9mg/100mlsVitreous4mg/100mls

Methanol & Isopropanol2mg/100mls of each in Blood, urine and vitreous

Supports ante-mortem consumptionKetoneButCan be produced pm

What else do you want to know?Urine Glucose negativeVitreous glucose unrecordable

Urine Ketones presentBlood Betahydroxybutyrate 2.3 mmol/L (

Case 4Decomposed @ home on sofa with drug paraphenalia around

LIVERHomogenate

Ethanol0.88 mg/gAcetoneNot detectedMethanolNot detected IsopropanolNot detected

General drug screen by gas chromatography mass spectrometry (GC-MS)Liver homogenate:Morphine, cocaine metabolites, flupenthixol and metabolites, paracetamol, chlorpromazine metabolites and cotinine detected detected

Liver Tissue Homogenate Quantitative Analysis by Gas chromatography Mass spectrometry (GC-MS)Cocaine=

Was it worth doing if the concentrations mean nothing?

COMMENTSThe external examination showed an advanced state of decomposition with no evidence of injury to suggest involvement of another individual.

Within the limits imposed by the degradation of the tissues, the internal examination showed no apparent pathology which could have caused or contributed to death.

Interpretation of toxicological results is complicated in cases where the individual has been dead for some time. Drug levels are altered after death by diffusion of the substances throughout the body (post-mortem redistribution) and certain substances are produced or degraded in the post-mortem period. As such, it would be unreliable to draw conclusions from the drug concentrations themselves. However cocaine and morphine are not produced endogenously by the body and so their presence indicates that cocaine and morphine (possibly heroin) were taken by the deceased. Alcohol can be produced by the body after death but the presence of cocaethylene would suggest that alcohol and cocaine were used concurrently.

The exact levels of the fore-mentioned drugs within the body at the time of death cannot be determined with any accuracy. However, we feel that their presence, along with the drug paraphernalia noted in the vicinity of the body and the absence of identifiable natural disease is significant. Based on the above information, we are of the opinion that, on the balance of probabilities, death was in keeping with polydrug toxicity.

The information given within this report represents our understanding of the views, opinions and circumstances of this case based on the information that we have received to date, either in writing (all forms) or by oral communication. We recognise that in part this may reproduce or rely upon witness statements, oral communications or hearsay evidence of second parties and that the information given to us by others may or may not be factually correct at the time of our consideration.We reserve the right to reconsider any aspect of this report should further factual information arise that contradicts the information provided at the time of the post-mortem examination, upon which we have based our interpretations.

Cause of DeathIa. In keeping with polydrug toxicity

ConsiderationsAsk YourselfArterial vs venous variationContinuing gastric residue absorptionRedistributionPost-mortem production or degradationToleranceLimited reference range dataWhere was it from?Vitreous / blood / urineSite

Was it appropriately preservedIs there decomposition?Is it a drug prone to redistribtution / pm productionDo you know about their drug taking / drinking habits

Confounding Factors

In PracticeSeek a drug history from coroners officerAlways give the toxicologists as much info as you haveIf you are looking for a specific substance tell them as it might not be on their routine screenAppropriate specimens / appropriate siteIf tox is important talk to coroners officer about accessing ante-mortem bloods.

Resourceshttp://www.dundee.ac.uk/forensicmedicine/C. Baselt, Disposition of Toxic Drugs & Chemicals in Man.