Guide to Obtaining Specimens at Post-mortem for Analytical Toxicology
Copyright ©
[Regional Laboratory for Toxicology]. All rights reserved.
1. INTRODUCTION
These guidelines have been written in order to
assist Pathologists in the selection of appropriate specimens of body fluids
and tissues for post mortem biochemical and toxicological analysis to be
carried out at the Regional Laboratory for Toxicology. More detailed
guidelines for pathologists have been published elsewhere (Forrest 1993;
Knight, 1996). It is particularly important to obtain samples from the
anatomical sites stated in the following guidelines. This will assist in the
interpretation of the results by reducing the problems caused by post mortem
drug distribution.
In
addition to indicating the exact site of specimen collection, it is also
important to provide any relevant available details regarding the medical and
drug history of the deceased as well as the circumstances of the death. The
Request for Post Mortem Toxicology Investigation form should be completed in
full (see Documentation).
Following death there can be
rapid changes in cellular biochemistry as autolysis proceeds, and drugs and
other poisons may be released from their binding sites in tissues and major
organs, also unabsorbed drug may diffuse from the stomach. Special care should
always be taken in the selection of blood and tissue sampling site(s), the
method of collection of samples, and the labelling of sample containers. There
is substantial published evidence to show that for most drugs and poisons,
including alcohol, there are important differences in their concentration in
blood according to the time of specimen collection after death, choice of
sampling site, method of sampling and volume of blood collected (Pounder and
Jones 1990; Pounder 1993). It is common to observe tenfold differences in the
concentration of certain drugs and some chemical poisons in post-mortem blood
taken from different sites. Specimens taken from "central" sites e.g. heart
tend to give particularly "high" values for most analytes. Moreover, certain
commonly used "peripheral" sites such as subclavian, may sometimes give
results closer to "central" sites such as the heart. The most consistent
quantitative findings are obtained in blood taken from the femoral vein, which
is the recommended site of specimen collection. It is also possible to observe
differences in the concentration of certain drugs obtained from different
tissue sampling sites for liver and lung.
2. COLLECTION OF URINE
AND BLOOD SAMPLES
The standard procedure for toxicological
analysis performed at this laboratory requires the collection of blood and
urine samples. Appropriate tissue (brain, liver etc.) and stomach contents
should be collected at post mortem but will not normally be required by this
laboratory unless special investigations are required; however they should
be retained at the mortuary. It is preferable to send specimens to the
Laboratory via the Coroner's Officer or via a reputable Courier service.
This is particularly important in certain medico-legal cases where, in order
to maintain the chain of custody, specimens should be submitted in person to
the Laboratory by the authorised Coroner's Officer. However, if this is not
possible, most specimens, particularly blood and urine, may be sent by post
if tightly sealed in their respective containers and securely packaged.
3. SUPPLY OF SPECIMEN
CONTAINERS
Depending upon stock availability the
Laboratory is able to provide appropriate specimen containers for the
collection of blood and urine specimens at post mortem, where cases are
submitted to this Laboratory for investigation. Please contact the
Laboratory if this service is required. In general, use containers
appropriate to the specimen volumes with secure closures. Plastic universal
(20 mL) containers are usually sufficient in addition to 5 mL fluoridated
glass containers.
NEW POSTAL REGULATIONS
From 1st January 2007 the Royal Mail
introduced new regulations for sending biological specimen through the post.
At the moment we have been informed that these guidelines are for infectious
samples and do not apply to specimens for diagnostic assays such as
toxicology screening. However, as many specimens received by this Laboratory
have a risk of infection, it is believed that these regulations will apply
to anybody sending post mortem specimens by post. We have not been informed
of a specific date as to when these regulations will be strictly enforced.
However, after such a date any package deemed unacceptable by the Royal Mail
will be refused delivery. We are carefully monitoring this development and
will hopefully be in a position to advise further regarding the exact nature
of these implications and address any problems that may arise.
Please contact the Laboratory for further
information.
4. LABELLING AND STORAGE
OF SAMPLES
All specimen bottles should
be clearly labelled with the full name of the deceased, date of collection,
the type of specimen and post mortem or reference number. In the case of blood
specimens, the specific site of sampling should always be given. All specimens
should be stored at 4oC before transporting them to the Laboratory. Each
specimen bottle should be securely sealed to prevent leakage, and individually
packaged in separate plastic bags to ensure that there is no
cross-contamination. Special care is required in the storage of specimens for
the analysis of certain poisons - please contact the Laboratory for guidance
if unsure.
Urine specimens
Urine specimens, however
small, taken at post mortem are of great value in screening for an unknown
drug or poison, particularly substances of abuse. If only a small amount of
urine is available, this should be placed in a plain 5mL glass tube.
Preferably at least 20mL of urine should be placed in 1-2 plain 20mL sterile
plastic container(s). Boric acid containers should NOT be used. Urine is also
valuable as a complimentary specimen in the quantitative analysis of alcohol,
where there is uncertainty over the validity of a blood specimen. For this
purpose, a urine specimen should be placed in a 5mL 1% fluoride/oxalate glass
tube.
Importance of site and method of sampling
The sites and methods of blood sampling are
important; care taken at this stage will often be rewarded by more reliable
and confident interpretation of toxicological findings. The concentrations of
some drugs, notably common compounds such as tricyclic antidepressants and
dextropropoxyphene, are much more likely to be “falsely” elevated in blood
from thoracic and abdominal vessels than in "peripheral" vessels such as
femoral veins. Moreover, even blood from these sites is easily contaminated by
blood drawn from contiguous, more central vessels if large specimens are taken
without ligation. The procedure recommended below for the collection of
femoral venous blood should minimise these effects.
Our current recommendation for an ideal set of
blood specimens for toxicological investigations is as follows:
Blood for quantitative analysis
Blood for quantitative analysis (~ 5mL) should
be obtained from two distinct peripheral sites, preferably left and right
femoral veins, taken with care so as not to draw a large volume containing
blood from more central vessels. The precise sampling site must be indicated
on the label. Femoral blood can be taken by cutting the external iliac vein
proximal to the inguinal ligament and milking the distal cut end into a plain
20mL sterile plastic container. Approximately 5mL of this blood should be
placed in a fluoride/ oxalate tube containing 1% (w/v) sodium fluoride as a
preservative.
Blood for qualitative analysis
(screening)
An
additional larger specimen of blood (~ 20mL) for qualitative screening should be
taken from the heart (preferably right atrium or inferior vena cava) or if
necessary from another convenient large vessel. This specimen should be placed
in a plain 20mL sterile plastic container. The site of collection must be
indicated on the label.
For deaths which have occurred in hospital, the
hospital pathology laboratory should be contacted as soon as possible to see
if any ante mortem specimens of urine, blood, serum, or plasma are available,
and these should also be sent for analysis. The exact date and time of
collection should be confirmed and indicated on the submission form. It is
also important to note if any antidotes, or drugs used in resuscitation were
given and if urine specimens were taken with the use of a catheter and/or
lignocaine local anaesthetic.
Other types of specimen will not normally be
required, but they may be valuable in the investigation of certain types of
poisoning and should be retained at the mortuary.
All organs should be placed in separate sample
containers to remove any chance of cross-contamination. A preservative such as
formalin must not be used. Sample containers should be clearly labelled.
Stomach tissue and contents
These materials may be useful in the
investigation of oral cyanide poisoning, or in cases of rapid death where
relatively large amounts of unabsorbed drug may be found in the stomach. In
cases of suspected drug overdosage the entire stomach contents should be
retained. If distinct tablets or capsules are observed in the stomach
contents, these should be carefully extracted, and put in individual
containers (e.g. plastic urine containers). Identification of such material
can be carried out by reference to a computerised database of pharmaceutical
products, or by direct analysis at the Laboratory.
Liver
This tissue may be useful in certain complex
poisoning cases. It is usual to take a portion of the right lobe of liver
since it should be uncontaminated with bile and less affected by drug
diffusion from the stomach; 100 grams are sufficient for most analytical
purposes.
Brain
A portion of about 100g brain may be useful
in the investigation of death due to gases or volatile substances (e.g.
butane). The specimen should be placed in a glass specimen jar or nylon bag
and deep-frozen prior to transport to the laboratory.
Lung
A portion of about 100g lung from the apex may
be useful in the investigation of death due to gases or volatile substances.
The specimen should be placed in a glass specimen jar or nylon bag and stored
at 4oC prior to transport to the Laboratory.
Hair
Hair specimens may be useful in the
investigation of death related to drug abuse (particularly opiates and
methadone). Analysis of hair (approximate rate of growth 1 cm per month) is
able to provide useful information concerning the chronicity of drug abuse,
which is valuable in the interpretation of post mortem drug concentrations. If
hair specimens are cut from the head, the proximal end should be clearly
identified; the cut end tied with a piece of thread. However, the Laboratory
does not currently provide a hair analysis service.
Vitreous Humour
A sample of vitreous humour is useful where a
body has been exposed to heat, or if putrefaction is beginning to occur. This
specimen may be especially useful for certain biochemical tests such as urea,
creatinine, glucose, lactate and electrolytes; in addition to analysis of
alcohols and heroin. More information on the collection of vitreous humour can
be found in the recent reviews of Forrest (1993) and Knight (1995, 2002).
Nails and Bone
These specimens may be
useful in the investigation of certain types of chronic metal
poisoning, notably arsenic and lead.
5. ADDITIONAL ADVICE
CONCERNING INVESTIGATION OF CERTAIN POISONS
Alcohol (ethanol)
Where there has been
evidence of putrefaction or extensive injury to the body or
there is a gap of several days between death and post-mortem,
it is advisable to take both blood and urine specimens for
alcohol analysis. In certain cases it might also be advisable
to take a specimen of vitreous humour. Ethanol can sometimes
be lost or generated from blood specimens if they have become
contaminated by bacteria or fungi. Use of specimen containers
containing 2% fluoride is therefore recommended for alcohol
measurement in "sensitive" cases.
If should be noted that
some enzymic methods for alcohol analysis commonly used by
clinical laboratories may give falsely elevated
results in critically ill patients or post-mortem specimens.
Analysis for alcohol performed by the Regional Toxicology
Laboratory is carried out by gas chromatography, which is
more specific than enzymic methods of alcohol analysis and is
also able to detect the presence of other alcohols such as
methanol and isopropanol, or the products of keto-acidosis
such as acetone.
Cyanide
In suspected cyanide
poisoning it is helpful to collect specimens of blood from
more than one peripheral site, and also stomach contents. In
cases where the source of cyanide is not known it may be
useful to obtain a small specimen of brain (~ 20 grams) from
a site deep within the brain to confirm the presence of
cyanide.
If there is prolonged
storage of blood specimens after post-mortem, it is possible
to generate significant quantities of cyanide, probably as a
product of bacterial action. The use of blood specimen
containers containing 2% sodium fluoride is recommended to
prevent this. Blood and tissue specimens are best stored
at 4oC and should be analysed as soon a possible
after collection. It is important to try to identify the
exact source of the cyanide taken by the deceased.
Carbon Monoxide
In cases involving
inhalation of vehicle exhaust fumes it is helpful to know the
make, year and model of the vehicle concerned. Many modern
petrol and diesel cars and other vehicles have catalytic
converters, which greatly reduce the amount of carbon
monoxide produced. Death from carbon monoxide poisoning may
be greatly delayed or impossible in certain circumstances.
Fire deaths
Measurement of both carbon
monoxide and cyanide may be helpful in cases involving fires.
However, analysis of blood specimens should be carried out
without delay. In such cases there is a tendency for the
carbon monoxide concentration to decrease with time
and the cyanide concentration to increase. Use of
blood specimen containers containing 2% sodium fluoride is
advisable, particularly in "sensitive" cases or
those involving multiple fatalities.
DRUGS & POISONS
Heroin
Heroin is a widely abused drug that is
involved in numerous deaths. However, there are difficulties when
investigating such deaths. In particular, as heroin (diacetylmorphine) is
rapidly metabolised to morphine it can be difficult to distinguish between
morphine and heroin overdosage. The first phase metabolite of heroin,
6-monoacetylmorphine (6-MAM), can be used as a "marker" for heroin but it is
rapidly metabolised and is unstable in blood. However, recently published
literature suggests that this compound may be more stable in the
cerebrospinal fluid (CSF) and vitreous humour. Therefore when investigating
deaths where heroin is involved/suspected it may be useful to obtain a
sample of the vitreous humour in addition to urine specimens and blood
specimens from both a "peripheral" and "central" site such as the femoral
vein and the heart, respectively.
Cocaine
Cocaine is another abused drug which is
relatively unstable in blood due to the presence of viable metabolically
active enzymes even in post mortem blood. Therefore, it is recommended that
in cases where cocaine ingestion is suspected, blood specimens should be
placed in fluoride/oxalate tubes containing at least 1% sodium fluoride. It
is also advisable to store the specimens at 4oC immediately after collection
and before delivery to the Laboratory.
Nitrazepam
Published data has shown that the
benzodiazepine, nitrazepam is also unstable in blood and therefore traces of
nitrazepam may be lost if the blood specimens are not preserved. In order to
do this we advise that such blood specimens should be frozen upon collection
at post mortem. Specimens should remain frozen prior to delivery to the
Laboratory. Generally, in cases of overdosage, nitrazepam can still be
detected in the absence of such treatment; however, the concentration will
inevitably be lower to some degree making interpretation difficult.
6. BIOCHEMICAL
INVESTIGATIONS
Biochemical investigations carried out on
post mortem blood are generally of limited value. Biochemical analysis of
vitreous humour can sometimes be useful but the interpretation of potassium
and sodium concentrations is complex. The presence of acetone and
measurement of b-hydroxybutyrate (BHB) in blood and urine may be an
indication of alcoholic or diabetic ketoacidosis and can provide useful
evidence particularly in sudden alcoholic deaths. The measurement of
glycated haemoglobin (HbA1c) is a much more reliable guide to
undiagnosed diabetes mellitus or elevated blood glucose concentrations over
a period prior to death and may be performed by hospital clinical chemistry
departments. If the injection of an insulin overdose is suspected, post
mortem blood and even vitreous humour may not be suitable for subsequent
analysis of insulin and C-peptide. Due to the instability of these
products and the lack of any comparative data, the interpretation of the
findings can be very difficult. Such analyses are not performed at the
Regional Laboratory for Toxicology. For cases where lithium may be
involved (e.g. the patient had been prescribed lithium), it is also
necessary to obtain a serum specimen as the measurement of lithium in post
mortem blood can present problems for both analysis and interpretation.
The Laboratory uses a wide range of modern
methods to analyse biological specimens for the presence of drugs and
poisons. Including:
· High performance liquid
chromatography (HPLC)
· HPLC with UV diode array
detection (HPLC-DAD)
· Gas chromatography (GC)
· GC with mass spectrometry
(GC-MS)
· Inductively coupled plasma mass
spectrometry (ICP-MS)
· Enzyme Multiplied Immunoassay
Technique (EMIT)
· Fluorescence Polarisation
Immunoassay (FPIA)
· UV Spectrophotometry
· Radioimmunoassay (RIA)
7. ANALYSIS
If should be noted that some enzymic methods
for alcohol analysis commonly used by routine clinical laboratories may give
falsely elevated results in critically ill patients or post mortem
specimens. Analysis for alcohol performed by the Regional Laboratory for
Toxicology is carried out by gas chromatography, which is more specific than
enzymic methods of alcohol analysis and is also able to detect the presence
of other alcohols such as methanol and isopropanol, or the products of
ketoacidosis such as acetone.
In the majority of cases, the investigation
will be carried out within 10 working days and an interim or final report,
including interpretation, will be issued within two weeks. For many
uncomplicated cases such as alcohol or carbon monoxide poisoning, a verbal
report will generally be available within 24 hours and written report within
5 working days. If there is likely to be a special problem with a particular
investigation, e.g. poisoning with recently introduced drugs, the Laboratory
will discuss the case with the Coroner's Officer or Pathologist.
8.
INTERPRETATION OF FINDINGS
Interpretation of findings
can present a problem where there is little background information
concerning the case, or where specimen collection has been inadequate. It is
difficult to provide any valid comment on the significance of quantitative
measurements carried out on a single blood specimen from an unknown site.
Interpretation of findings can also be difficult in drug abusers where the
likely degree of "tolerance" to a drug is unclear because of inadequate
history. In addition, much of the published literature on forensic
toxicology relating to so-called "fatal" blood concentrations can be
misleading in certain circumstances. It is a common misconception that the
concentration of a drug (or poison) found in post mortem blood is equivalent
to that obtained in the blood or plasma of the deceased at the time of
death. Interpretation of findings will always need to take account of
changes in drug distribution after death. The recent history, age and state
of health of the deceased are also important factors to be taken into
account in the interpretation of findings. The Laboratory is always happy
to discuss individual cases or give further advice on the interpretation of
findings.
9. DOCUMENTATION
The following documentation is
desirable in every case:
· Details of the deceased, including
name, age and date of birth.
· Relevant medical history,
particularly with regard to prescribed medication and whether the deceased
suffered from a serious infectious disease such as hepatitis, tuberculosis or
AIDS.
· Circumstances surrounding the death,
including date/time of death if known (particularly regarding deaths in
hospital).
· Details of the name and quantity of
the substance(s) thought to have caused death.
· A copy of the Pathologist's
preliminary report indicating the likely cause of death.
· Details of the type of specimens
obtained and the specific site of collection.
· Name, address and telephone number of
the Pathologist and Coroner's Officer involved in the case.
· State HM Coroner's District and name,
address and telephone number of HM Coroner (for billing purposes).
All of the above information is required to
enable the Laboratory to provide an accurate interpretation of results and an
efficient service.
Request forms for toxicological analysis are
available from the Laboratory
10. TRANSPORT OF
SPECIMENS
Most specimens,
particularly blood and urine, may be sent by post if securely
packaged. In certain medico-legal cases, in order to maintain
the chain of custody, specimens should ideally be submitted
in person to the Laboratory by the authorised Coroner's
Officer.
13. RECOMMENDED
READING
Knight, B. (1996),
Forensic Pathology
(2nd edition)
Arnold, London.
Knight, B. (2002),
The estimation of the time since death in the
early postmortem period (2nd edition)
Arnold Publishing, London.
Pounder, D.J. and Jones, G.R.
(1990),
Post-mortem Drug Redistribution
- A Toxicological Nightmare,
Forensic Sci. Int. 45
: 253-363.
Forrest, A.R.W. (1993),
Obtaining samples at
post-mortem examination for toxicological and biochemical
analysis,
J. Clin. Pathol. 46
: 292-296.
The Hospital Autopsy (1993),
D.W.K. Cotton & S.S Cross
eds.
Butterworth, Heinemann, Oxford.
Pounder, D.J. (1993),
The Nightmare of Post-mortem
Drug Changes
In: Legal Medicine
Butterworth Legal Publishers,
Salem, New Hampshire
pp. 163-191.
For more information or
guidance please contact:
Dr S.P. Elliott,
Senior Clinical Scientist, Forensic Toxicology Section Head,
Telephone 0121 507
5204 (Direct line).
E-mail
simon.elliott@swbh.nhs.uk
Fax No. +44 121 507 6021
Revised: May 2004
Last edited:
May 31, 2007
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