Accredited Medical Laboratory, Reference No: 0814 FORENSIC TOXICOLOGY SERVICE LEICESTER ROYAL INFIRMARY USER HANDBOOK 2016-17 1 of 12
CONTENTS Contacts and normal laboratory hours 3 Our services 4 Specimen collection 4 Specimen labelling 7 Specimen transport 7 Requests for analysis 8 Analysis 9 Quality control 10 External quality assurance 10 Interpretation of analytical findings 11 Report distribution 11 Specimen storage 11 Turnaround times 11 Urgent requests 1 Costs for analyses 12 2 of 12
FORENSIC TOXICOLOGY SERVICE UNIVERSITY HOSPITALS OF LEICESTER NHS TRUST LEVEL 4, SANDRINGHAM BUILDING LEICESTER ROYAL INFIRMARY LEICESTER, LE1 5WW Telephone Numbers & Enquiries Consultant Chemical Pathologist and Forensic Toxicologist Dr Stephen Morley Tel: 0116 258 5772 E-mail: steve.r.morley@uhl-tr.nhs.uk Consultant Clinical Scientist and Forensic Toxicologist Dr Paul Smith Tel: 0116 258 5772 E-mail: paul.r.smith@uhl-tr.nhs.uk Forensic Toxicology Administrator and Secretary Teresa Hincks Tel: 0116 258 5772 E-mail: teresa.l.hincks@uhl-tr.nhs.uk Fax: 0116 258 6550 Principal Biomedical Scientist Richard Cole Tel: 0116 258 7589 E-mail: richard.cole@uhl-tr.nhs.uk Principal Clinical Scientist Laura Hikin Tel: 0116 258 7589 E-mail: laura.hikin@uhl-tr.nhs.uk Normal Laboratory Hours Monday to Friday: 9.00am - 6.00pm (except on Bank Holidays) 3 of 12
OUR SERVICES The Forensic Toxicology Service was established in collaboration with Home Office Pathologist Professor Guy Rutty MBE in 2001 to provide clinical and forensic toxicology services to HM Coroners, Home Office Pathologists, Consultant Histopathologists, police forces and other external laboratories. We have over 14 years experience in providing post mortem toxicology services to HM Coroners and currently provide services to over 20 Coroners. In addition, the laboratory provides a forensic toxicology service for suspicious deaths investigated by Forensic Home Office Pathologists for a number of police forces including the five East Midlands police forces (Leicestershire, Nottinghamshire, Northamptonshire, Lincolnshire and Derbyshire) on behalf of the relevant HM Coroner. In addition to toxicological services we routinely perform biochemical analyses on both ante mortem and post mortem samples to assist in determining the cause of death. All samples are handled by the laboratory under full chain of custody conditions. In contrast to many other service providers, we are able to provide both clinical, biochemical and toxicological analyses with comprehensive interpretation in written reports and expert witness testimony at competitive rates and with rapid turnaround times. We also provide a Road Traffic Acts Analysts service to members of the public in which blood or urine samples are analysed for the presence of alcohol. This service includes the provision of a report in the form of a witness statement and expert witness attendance in court if required. We are a NHS laboratory with full Clinical Pathology Accreditation (CPA incorporating ISO 15189) and participate in national and international external quality control schemes to ensure our analyses are of a consistently excellent standard. Our laboratory is equipped with an extensive range of instrumentation and we are constantly updating and developing new methods to detect and quantitate novel medications and drugs of abuse including designer stimulants and synthetic cannabinoids. The highly qualified staff team in the department bring great expertise, experience and professionalism from both clinical and scientific backgrounds. SPECIMEN COLLECTION The Post Mortem Toxicology Request Form should be completed in full. It is important to provide any available relevant information regarding the medical and drug history of the deceased as well as the circumstances of the death, in addition to indicating the site of specimen collection. It is also important to state whether the deceased was administered therapeutically any resuscitation fluids or drugs prior to his/her death. 4 of 12
Ante mortem Samples Where an individual has died several hours or days after admission to hospital and drug toxicity is suspected at the time of admission to hospital, the local hospital laboratory should be contacted as soon as possible to determine whether or not any hospital admission samples (blood and urine) are available for analysis. Post mortem Samples SPECIMEN AMOUNT ANALYSES PERFORMED Plain blood 10-20 ml Specific drug screen, drug quantitation, beta-hydroxybutyrate, HbA1c, carboxyhaemoglobin, mast cell tryptase, insulin, C-peptide Preserved blood (tube containing a preservative) 5 of 12 5 ml Alcohols, acetone, cyanide, ethylene glycol, other volatile substances Plain urine 10-20 ml General drug screen Preserved urine 5 ml Alcohols, acetone (tube containing a preservative) Plain vitreous All available Urea, creatinine and electrolytes, specific drug screen, drug quantitation Preserved vitreous All available Alcohols, acetone and glucose (tube containing a sodium fluoride preservative) Stomach contents 10-20 ml Ethanol, general drug screen Liver tissue 50 g Specific and general drug screen, drug quantitation Lung tissue 50 g Volatile substances Brain tissue 50 g Volatile substances Hair Thickness of a pencil Specific and general drug screen, drug quantitation Please note that the specimen amounts indicated above are the ideal quantities to allow comprehensive analyses and to enable repeat analysis to be performed if required. Blood 10-20mL blood taken into a plain 20mL sterile plastic container is required for drug qualitative (screening) and quantitative analysis. Blood for quantitative analysis should preferably be collected from the femoral vessels to reduce the effects of post mortem redistribution. However, if this is not possible, please use other sites for collection. The site of blood collection should be stated on the specimen label and the request form. An additional 5mL blood should be collected into a separate tube containing sodium fluoride preservative. This inhibits bacterial metabolism of analytes such as ethanol and also prevents extensive degradation of labile drugs such as 6-monoacetylmorphine (6- MAM) and cocaine.
Urine 10-20mL plain and 5mL preserved (sodium fluoride) urine should be submitted to the laboratory. Urine is useful for ethanol analysis and also qualitative drug screening. Drugs and their metabolites are usually present in urine at much higher concentrations than in blood and are detectable for longer periods of time following ingestion. Vitreous Humour All vitreous humour should be collected into either a 5mL tube containing sodium fluoride preservative or a plain 20mL sterile plastic container. As vitreous is collected from a relatively contained compartment it offers a number of advantages over more commonly submitted specimens, particularly when some putrefaction of the body has occurred. Vitreous humour is particularly advantageous for confirmation of blood ethanol concentrations as it is well protected from bacterial infiltration and is, therefore, less prone to post mortem fermentation often observed in blood. Vitreous humour is also useful for biochemical tests such as glucose, urea, creatinine and electrolytes. Potassium may be measured to assist in the estimation of the post mortem period. Ethanol and glucose analysis is preferentially performed on a preserved sample, whilst sodium and potassium levels are measured on plain vitreous samples only as the preservative often consists of sodium (sodium fluoride) and potassium (potassium EDTA). Stomach Contents 10-20mL stomach contents should be collected into a plastic container without preservative. The total stomach content volume should be stated on the request form. This matrix is useful in cases of oral drug ingestion / overdose in which large amounts of unabsorbed drug may remain in the stomach. If recognisable tablets or capsules are found, these should be removed and placed in separate containers for identification. Ethanol analysis of stomach contents may also be performed to assist in the determination of the absorptive phase of ethanol. Liver Tissue Liver tissue is usually submitted in cases where little or no blood is obtained. Approximately 10g of wet unfixed tissue should be placed into a plastic container. As it is the primary metabolic organ, drugs are often found in higher concentrations than in the blood. Data suggest that concentrations can vary depending on the site from which the liver specimen is collected. It is recommended that the right lobe be sampled to reduce contamination from bile and drug redistribution from the stomach contents. Brain Tissue A portion of approximately 10g brain may be useful in the investigation of death due to gases or volatile substances (see page 9, volatile substances). The specimen should be placed into a plastic plain histology container. 6 of 12
Lung Tissue A portion of approximately 10g lung from the apex may be useful in the investigation of death due to gases or volatile substances (see page 9, volatile substances). The specimen should be placed into a plastic plain histology container. Other Tissues Other solid tissues such as muscle may be used for drug analysis. However, there is limited published peer reviewed literature on the interpretation of these results. Hair Hair analysis provides a large window of detection and an accurate retrospective record of drug use. It takes 3-6 days following drug use for the affected hair to emerge from the scalp and hair grows at a rate of approximately 1 cm per month. Ideally a minimum of 100mg of hair should be cut from the back of the head of the deceased. The root and tip ends of the submitted hair specimen should be clearly marked. Due to the complex and labour intensive nature of hair analysis an additional charge is applied, which is dependent upon the length of the hair sample and the number of drugs that require quantitation. SPECIMEN LABELLING All specimen bottles should be clearly labelled with the full name and date of birth of the deceased (if known), date of sample collection, specimen type, and the mortuary reference number or exhibit number (if applicable). As mentioned earlier, in the case of blood specimens, the specific site of sampling should also be stated. Each specimen bottle should be securely sealed to prevent leakage and placed into sample bag(s) that separate the request form from the samples. All specimens should be stored at 4 C prior to delivery to the laboratory. High risk samples must be clearly labelled. SPECIMEN TRANSPORT A specimen collection service under full chain of custody conditions from the mortuaries at Leicester Royal Infirmary and Queen s Medical Centre, Nottingham is provided by the laboratory. However, in certain medico-legal cases specimens may ideally be submitted in person to the laboratory by the Pathologist, Police or authorised Coroner's Officer in order to maintain the chain of custody. Where samples are sent by post we recommend that they are sent by Special Delivery or Courier Service to ensure that chain of custody is maintained throughout. 7 of 12
REQUESTS FOR ANALYSIS Alcohol Analysis Ethanol and the presence of other alcohols (methanol and isopropanol) are routinely quantitated by head space-gas chromatography (HS-GC) in blood, urine and vitreous. Ethanol analysis in stomach contents may also be determined to assist in the interpretation of the absorptive phase of ethanol. Methanol, isopropanol and acetone levels are also determined by HS-GC. Ethylene glycol is a toxic alcohol often used in engine antifreeze or as a radiator coolant. Blood ethylene glycol levels are measured in cases of suspected ethylene glycol ingestion and this should be stated on the request form to allow a specific GC assay to be performed. Alcohol and acetone analysis is preferentially performed on preserved samples. Drug screening A specific drug screen by liquid chromatography-tandem mass spectrometry (LC- MS/MS) in blood for over 400 of the most commonly prescribed and abused drugs is routinely performed. A general drug screen by gas chromatography-mass spectrometry (GC-MS) is also routinely performed on urine, which detects the vast majority of drugs/medications and also 'unknown' drugs such as certain new psychoactive substances. If no blood or urine specimens are available drug screening may be performed on vitreous humour or liver tissue samples. Drug quantitation The majority of drugs detected are quantitated by LC-MS/MS, isotope dilution GC-MS or high performance liquid chromatography (HPLC). Specific Analyses Biochemical Investigations The concentrations of urea and creatinine change little over time, which means that it is possible to use these measurements to make post mortem diagnosis of renal failure and/or severe dehydration. Sodium concentrations are thought to decline in the post mortem period depending on the variability of body storage conditions. Potassium concentrations increase after death and may therefore be measured to assist in the estimation of the post mortem interval (time since death). The presence of acetone in blood, urine and vitreous and the measurement of betahydroxybutyrate in blood may be used in the diagnosis of alcoholic or diabetic ketoacidosis. The measurement of glycated haemoglobin (HbA1c) in blood provides a reliable indicator of the long term glycaemic control in an undiagnosed or known diabetic. Vitreous humour glucose concentrations tend to fall very quickly after death and a low concentration does not necessarily indicate ante-mortem hypoglycaemia. If an insulin overdose or hypoglycaemia is suspected, post mortem serum may be suitable for the analysis of insulin and C-peptide. However, the interpretation of the findings can be 8 of 12
very difficult due to the instability of these compounds and the unreliability of post mortem glucose levels. Blood and bile spot acyl carnitine, and urinary organic acid levels are measured to assist in the metabolic investigation into the cause of sudden unexplained death in infancy. In the absence of a sufficient urine sample to perform organic acid analysis, a bladder tissue wash is used to generate a sample for analysis. Carboxyhaemoglobin (carbon monoxide) and cyanide Carboxyhaemoglobin levels are measured in blood in cases of suspected carbon monoxide poisoning and smoke inhalation from all types of fires. Hydrogen cyanide which is produced in fire smoke from many synthetic polymers containing nitrogen or halogens may also be measured in blood. Immunological Investigations Mast cell tryptase is a constituent of mast cell granules and is released together with histamine in specific allergic reactions mediated by IgE. Mast cell tryptase levels may be elevated following an anaphylactic episode. The measurement of total IgE levels is not recommended in post mortem blood. Volatile Substances Volatile substances can be classified according to their chemical structure, i.e. aliphatic hydrocarbons (e.g. propane, butane and isobutane), aromatic hydrocarbons (e.g. benzene), oxygenated compounds (e.g. ether) and halogenated compounds (e.g. chloroform). The components of natural gas include methane, ethane, propane and butane. After being taken up from the lungs into blood, volatile substances are distributed at high concentrations into lipid-rich tissues such as the brain, fat tissues, liver, heart and kidney. Therefore, in addition to a preserved blood sample, it is recommended that lung and brain tissue samples should be submitted for the investigation of volatile substance deaths. ANALYSIS The laboratory uses a wide range of modern analytical methods to analyse ante mortem and post mortem specimens for the presence of drugs and poisons including: Head space gas chromatography with flame ionisation detection (HS-GC-FID) Gas chromatography with flame ionisation detection (GC-FID) Gas chromatography-mass spectrometry (GC-MS) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) HPLC with diode array detection (HPLC-DAD) Enzyme immunoassays Spectrophotometry Inductively coupled plasma mass spectrometry (ICP-MS) Fourier transform infra-red spectroscopy 9 of 12
QUALITY CONTROL (QC) Internal QC is used to check the validity of analytical results on a day to day basis. QC material is either purchased from a recognised commercial source (e.g. Chromsystems, LGC, Biorad or UTAK) wherever possible or prepared in-house if appropriate. EXTERNAL QUALITY ASSURANCE (EQA) The laboratory participates in national EQA schemes to monitor the accuracy and precision of its analyses including: LGC Heathcontrol External Quality Assessment Scheme for Toxicology Case Studies in serum and urine This scheme covers the analytical investigation of a toxicological case and the subsequent interpretation of the results obtained in relation to the case history given. This laboratory is a member of the advisory and scoring panel for this scheme. LGC Quartz Post Mortem Blood Toxicology Proficiency Testing Scheme This scheme provides for the identification and quantitation of drugs in forensic toxicology samples. United Kingdom National External Quality Assessment Service (UK NEQAS) Scheme for Analytical Toxicology in blood, serum and urine This scheme covers ethanol, paracetamol, salicylate and other drugs. UK NEQAS Scheme for Drugs of Abuse in urine This scheme covers a minimum of amphetamines, barbiturates, cannabinoids, cocaine, benzodiazepines, methadone/eddp, opiates (including 6-MAM), buprenorphine and metabolites, MDMA, MDA and MDEA in addition to measures of sample integrity. Heath Control Scheme for Therapeutic Drug Monitoring in serum This scheme covers carbamazepine, clonazepam, digoxin, ethosuximide, lamotrigine, lithium, phenobarbitone, phenytoin, primidone, theophylline, valproate, clobazam, oxcarbazepine, gabapentin, levetiracetam, vigabatrin, amiodarone, flecainide. Heath Control Scheme for Antipsychotic drugs in serum This scheme covers amitriptyline, nortriptyline, doxepin, fluoxetine, fluphenazine, sertraline, trimipramine, risperidone, mirtazepine, imipramine, desimipramine, clomipramine, dothiepin, maprotiline, venlafaxine, thioridazine, haloperidol, fluvoxamine, perphenazine, quetiapine, amisulpiride and citalopram. 10 of 12
INTERPRETATION OF ANALYTICAL FINDINGS Interpretation of our analytical findings is provided in our comprehensive forensic toxicology reports and witness statements. Our interpretation takes into account numerous factors including drug reference ranges, post mortem drug redistribution, development of drug tolerance, post mortem drug metabolism (formation / breakdown), drug-drug interactions, site-to-site variability in drug levels and pharmacogenetics. It is therefore important to provide any available relevant details regarding the medical and drug history of the deceased as well as the circumstances of the death in addition to indicating the site of specimen collection. The laboratory is available for advice and to discuss specific cases. REPORT DISTRIBUTION Signed copies of the forensic toxicology report / witness statement are issued to the requesting Pathologist and HM Coroner and, when applicable, to the Police by secure e- mail. Paper copies of reports are available upon request. SPECIMEN STORAGE Samples submitted by a Consultant Pathologist on behalf of HM Coroner are stored under full chain of custody conditions for six months following the issue of a forensic toxicology laboratory report. Samples submitted by a Home Office Pathologist on behalf of HM Coroner or police are stored under full chain of custody conditions until the laboratory is notified that the case is closed or the samples are no longer required. Sample disposal is performed in a respectful manner in accordance with the Human Tissue Act 2004. TURNAROUND TIMES For the majority of uncomplicated cases that require no drug quantitation a forensic toxicology report or witness statement is usually available within 5-10 working days. For more complex cases that require drug quantitation a full report or witness statement including full interpretation is usually available within 10-20 working days. If there is a highly complicated case that is likely to be delayed due to the nature of the particular investigation/s (e.g. poisoning with recently introduced drugs) the laboratory will discuss the case with the relevant HM Coroner / Pathologist or Police and, if appropriate, issue a preliminary report. A final report is subsequently issued upon completion of all toxicological analyses. URGENT REQUESTS Please contact the laboratory to arrange for a case to be processed with a higher priority. 11 of 12
COSTS FOR ANALYSES The cost of analysis is dependent upon the analyses performed and also the workload submitted to the laboratory. Please contact the laboratory for further information. 12 of 12