DR A J JEFFERY

MBChB MD FRCPath (Forensic) MFFLM

HOME OFFICE REGISTERED FORENSIC PATHOLOGIST

Post-mortem Toxicology

Areas to Cover

Why 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 ?

Samples

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

Fluoride – inhibits further alcohol production but won’t undo the damage already done.

VitreousStomach Contents

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

OBTAINING THE

BLOOD SAMPLE

Femoral Vein Sampling

Vein NOT arteryBefore eviscerationBefore urine sampling

Ideal = tie off / clamp, then sample by wide-bore needle below

Routine = clean catch

Ideal = don’t milk the legRoutine = required to gain sufficient sample

MINIMAL FEMORAL BLOOD

Problem:

Insufficient Femoral Blood

Take what ever you can in preserved tubes Subclavian = 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 Sampling

Needle and syringe ororOpen 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?

Analysis

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

Confirmation (GCMS) Specific drugs found by breaking them down &

looking at the breakdown products.

Quantification Technique may vary dependent on the nature of the

drug being analysed.

HOW TO INTERPRET

THE RESULTS

General Considerations

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

Accuracy of reference ranges

Interpretation 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 Considerations

Re-distribution – site matters!Individual variation (renal disease)DecompositionTolerance

Redistribution

Discovered with digoxinMost drugs that undergo

redistribution do so because of their relative lipid solubility.

Due to Loss of cell integrity Diffusion

GI tract – to adjacent structures

Through conduits – lymphDiffusion from bladder

Natural Disease

Depends or route of administration 1st pass / second pass metabolism

Absorption With or without meal GI surgery

Elimination / Clearance Renal impairment Liver impairment

Decomposition

Significant redistribution

Some drug levels increase Alcohol production by bacterial action

Others degrade

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

Tolerance

Increasing doses required over time to achieve same effects.

What is lethal to a naïve 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 Alcohol

What alcohol related causes of death do you know?

How might you classify them?

Which specific toxicological causes do you know?

Alcohol

Acute alcohol toxicity

Ketoacidosis

Alcohol in combination with other drugs

Problems with Interpretation of Alcohol

Redistribution

Dealing with decomposition

Back calculations

Acute alcohol toxicity

How 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 skills 30 – 50 = deterioration in

driving 50 – 100 = inhibitions /

laughter 100 – 150 = slurring,

insteadiness, poss nausea 150 – 200 = obvious

drunkenness, nausea staggering

200 – 300 = stupor, vomiting, coma

300 + = 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 ½ 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?

Ketoacidosis

Brain 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 ketoacidosis Metabolic acidosis Malnutrition Binge drinking superimposed on chronic alcohol abuse

Ketoacidosis

Ketones: Acetone (can be produced pm)

<0.5 mg/100ml Beta hydroxybutyrate (less likely to be raised

artefactually) <0.5 mmol/L 1.26 – 47.2 mmol/L (assoc with fatalities)

Causes Alcoholic ketoacidosis Diabetic ketoacidosis

Alcoholic vs Diabetic

How might you differentiate?

Urine glucose

HbA1c 4 - 6.1%

Calculations

AVOID !

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

Widmark equation Used by some to predict amount of alcohol consumed

Decomposition

70 – 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 : Blood Vitreous : 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 opioids

MorphineHeroin (diamorphine) – IV, smoked, sniffedMethadone (green liquid – oral or IV)Pethidine, buprenorphine

** CNS depression **

Findings

History / scene / paraphernalia

External iv sites Foam at nose / mouth

Limited & non specific Pulmonary congestion and oedema Stomach contents – methadone is usu green!

Morphine / Heroin

Heroin / diamorphine – synthetic morphine derivative Powerful 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 hours Ie recent intake / top up injections

Acute alcohol intoxication potentiates the effects

Total morphine : Free Morphine Gives some idea of time since administration Eg in IV admin

15 mins post admin 4 : 1 60 mins 9 : 1

Therapeutic Lethal_______ Free morphine 10 – 100ng/ml 50 – 4000 ng/ml

Heroin

A contaminate of street heroin is acetylcodeine Hence may have +ve codeine levels

Most heroin deaths occur several hours after taking the drug Sleepy / snoring May have time to metabolise drug despite irreversible

respiratory depression

Methadone

Therapeutic 75 – 1100 ng/ml

Toxic 200 – 2000 ng/ml

Lethal 400 – 2000 ng/ml

Significant overlap Tolerance becomes very important Interpretation requires knowledge of drug history Long & variable T ½

Methadone

Breakdown product – EDDP This is inactive

Titration is important Many deaths occur during first few weeks of treatment Can cause respiratory depression at therapeutic doses

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

Opioids

Tolerance = V V important consideration Eg. Prison release Palliative care

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

TherapeuticLethal_______

Free Codeine 30 – 340ng/ml >1600 ng/ml

Sympathomimetics

Sympathomimetics

Incr activity of adrenaline and serotonin

Adrenalin Hypertension Tachycardia Mydriasis

Serotonin Excitement Hyperthermia

Stimulants

CocaineAmphetamineEcstasyOther methamphetamines

Associated with subarchnoid haemorrhage 80% of these assoc with aneurysms

Intracerebral haemorrhage Associated with AVMs & hypertension

Findings

Hearts of stimulant users tend to be heavier than controls

FibrosisContraction band necrosisAccelerated atherosclerosisNon specific pulmonary changes

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

Cocaine

Naturally occurring plant alkaloid stimulantSnorted, smoked, cutaneous, injectedNb always consider in sudden death in the

same way that you might consider HOCM.

Breakdown Products Benzoylecgonine Methylecgonine Cocaethylene

Inactive

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

Cocaine Toxicity

Less than 50 ng/ml cocaine is considered not to produce measurable physiological effects

T ½ can be as little as 40 minsBenzoylecgonine – 1-4 days in urine

Toxic >900 ng/ml

Lethal >1000 ng/ml

Benzoylecgonine Lethal - >1000 ng/ml *

Cocaine

But 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 crisis

Non 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 delirium

Excited delirium Hyperthermia Mental & physiological arousal Excited, erratic & sometimes bizarre, violent behaviour May have florid psychosis May exhibit extra-ordinary strength

Tends to result in sudden respiratory arrest Blood cocaine and benzoylecgonine may be low but there is

usually concentration of benzoylecgonine within the brain indicating long term use

Marked decease in D2 receptors in hypothalamus in psychotic cocaine abusers. D2 receptors play a role in temperature regulation

Amphetamine

Prevalence 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.

Amphetamine

Toxic >500 ng/ml amphetamine >1800 ng/ml methamphetamine

Lethal Usu > 1000 ng/ml amphetamine But can be seen if >50ong/ml Usu > 10 000 ng/ml methamphetamine

Different Forms

Amphetamine (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)

Amphetamine

Alcohol can potentiate effects on the heart.Rare toxic effects:

Coma Cerebral vasculitis Cerebral haemorrhage Rhabdomyolysis D.I.C. Renal dysfunction

Cardiac – long term users Accelerated coronary atherosclerosis Microvascular disease

MDMA / Ecstasy

Amphetamine-like drugs 3,4-methylenedioxymethamphetamine

Serotinergic and noradrenergic effects

• Hyperthermic effects

Liquid ecstasy – GHB Gamma hydroxybutyrate

Other

Benzodiazepines

Diazepam – 20 – 4000 ng/ml Nordiazepam – 20 – 1800 ng/ml

Need in the order of thousands to consider fatal.

Cannabis

Cannabinoids THC – tetrahydrocannabinol Delta 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 in Blood – up to ~ 5 days Urine – up to ~ 12-36 days

Volatile substance abuse - VSA

Adhesives, aerosols, petrols, paint stripper, nail varnish remover ….. amongst others

Increased risk taking behaviourAccidental suffocationCNS depressionDeaths thought to be due to cardiac arrhythmias

Sensitisation of myocardium to effects of adrenaline Deaths often seen in association with physical exertion

Blood 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’ Drugs

Mephadrone‘Cream’

IN

OTHER

CAUSES OF DEATH

Toxicology

Fire deaths

Carboxyhaemoglobin Normal <10% Toxic 15 – 35% Lethal >48%

Cyanide Normal < 0.1mg/L Develops within the potted blood sample if not

preserved

Carbon Monoxide

In an individual breathing air T ½ = 4 hours

Breathing O2 in Hospital T ½ = 60 minutes

Therefore always consider survival time.

Therapeutic Drugs

Anti-depressantsAnti-convulsants

Overdose

Aspirin Therapeutic

20 – 100mg/l Toxic

>150 mg/l Lethal

>500 mg/l

NB those on reg Rx (eg arthritis – 3g/day) – 44-330mg/L T ½ up to 36 hours in massive OD

Findings

Pm Blood stained gastric content / frank haematemesis Rarely skin petechiae Mucosal gastric erosions Malaena if survival sufficiently long Petechiae through other organs due to anticoagulant

effect esp parietal pleura and epicardium

Paracetamol Therapeutic

10 – 20 mg/l

Toxic >150 mg/l

Lethal >160 mg/l

IMMUNOLOGY – ANAPHYLAXISBIOCHEMISTRY – DIABETES

Other samples of interest

Immunology - Anaphylaxis

Secure 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 IgE

Make sure you give details of any suspected cause Available for venoms, foods, medicines, contrast agents,

latex…….

Mast cell tryptase

T ½ 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 activation High levels will be found post severe anaphylaxis Levels are not raised in local allergic reaction eg rhinitis Can be raised in pure asthma deaths but not of the same order of

magnitude Slight increases can be seen in

non-anaphylactic mast cell degranulation – EG opioids for chest pain Trauma – disruption of mast cell rich tissues

Unless grossly elevated – interpret with caution In presence of suggestive history and absence of pm findings it may provide

confirmatory evidence.

Normal Levels:

IgE 0 – 122 kU/L MCT 2-14 mg/L

MCT can be produced pm

Biochemistry - Diabetes

Vitreous is best for biochem as most blood is already haemolysed Glucose drops significantly after death

Bacterial metabolism Drops even in vitreous So low glucose ≠ hypoglycaemia

It 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

Insulin If 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 1

60 yr old male, in house fire, extensive burns, no suspicious injuries, no soot in airways or stomach. Ranges Norm Tox Leth CO = 40% <10% 15-35% >50%

Cyanide = 0.5 mg/L <0.1mg/L

Paracetamol 10-20mg/l >150mg/l>160mg/l 6mg/l

Codeine 30-340ng/ml >1600ng/ml 56ng/ml

Morphine (free) 10-100ng/ml 50-4000ng/ml <5ng/ml

Case 2

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

Ethanol Blood 181 mg/100mls Urine 244 mg/100ml

Ethylglucuronide present in urine

Acetone negative Methanol negative Isopropanol negative

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

Is this likely to by PM alcohol production?

Ranges: Normal Toxic Lethal

Methadone 75-1100ng/ml 200-2000ng/ml 400-2000ng/ml 413 ng/ml EDDP = 276 ng/ml

Amphetamine >500ng/ml >1000ng/ml 471 ng/ml

Diazepam 20-4000ng/ml >5000ng/ml >30 000ng/ml 21 ng/ml Nordiaz = 73ng/ml 20-1800ng/ml

Cause of Death??

Case 3

Chronic alcoholic found dead at home, head injury consistent with fall to the ground. Ethanol

Blood 16 mg/100mls Urine 72 mg/100mls Vitreous 25 mg/100mls Stom Cont 145 mg/100mls

Acetone Blood 3mg/100mls Urine 9mg/100mls Vitreous 4mg/100mls

Methanol & Isopropanol 2mg/100mls of each in Blood, urine and vitreous

Supports ante-mortem consumption

KetoneButCan be produced pm

What else do you want to know?

Urine Glucose – negativeVitreous glucose – unrecordable

Urine Ketones – present Blood Betahydroxybutyrate 2.3 mmol/L (<0.5 mmol/L)more specific than acetone

HbA1c – 12.1% (4.0 – 6.1%) Diabetes – reconsider urine alcohol

Urine ethyl glucuronide - present

Case 4

Decomposed @ home on sofa with drug paraphenalia around

LIVERHomogenate

Ethanol 0.88 mg/gAcetone Not detectedMethanol Not detected Isopropanol Not 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 = <0.02 ug/gBenzoylecgonine = 0.28 ug/g

Methylecgonine = 0.26 ug/gCocaethylene = 0.42 ug/gMorphine (free) = 1.05 ug/gMorphine (total) = 1.50 ug/g

Was it worth doing if the concentrations mean nothing?

COMMENTS

The 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 Death Ia. In keeping with polydrug toxicity

Considerations Ask Yourself

Arterial vs venous variation Continuing gastric residue

absorption Redistribution Post-mortem production or

degradation Tolerance Limited reference range

data

Where was it from? Vitreous / blood / urine Site

Was it appropriately preserved Is there decomposition? Is it a drug prone to

redistribtution / pm production

Do you know about their drug taking / drinking habits

Confounding Factors

In Practice

Seek a drug history from coroner’s 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 screen

Appropriate specimens / appropriate siteIf tox is important talk to coroner’s officer

about accessing ante-mortem bloods.

Resources

http://www.dundee.ac.uk/forensicmedicine/C. Baselt, Disposition of Toxic Drugs &

Chemicals in Man.