Dinis Oliveira 2010

Toxicology Mechanisms and Methods, 2010; 20(7): 363414
REVIEW ARTICLE
Collection of biological samples in forensic toxicology

R. J. Dinis-Oliveira1,2,3, F. Carvalho3, J. A. Duarte4, F. Remio3, A. Marques5,6, A. Santos1,5,6, and
T. Magalhes1,5,6,7
1
Faculty of Medicine, University of Porto, Porto, Portugal, 2Department of Clinical Analysis and Public Health, Center of
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Calgary on 10/07/12
Research in Health Technologies (CITS)-IPSN-CESPU, CRL, Vila Nova de Famalico, Portugal, 3REQUIMTE, Department
of Toxicology, Faculty of Pharmacy, University of Porto, Porto, Portugal, 4CIAFEL, Faculty of Sport, University of Porto,
Porto, Portugal, 5North Branch, National Institute of Legal Medicine, I.P., 6Center of Forensic Sciences, Portuguese Science
and Technology Foundation, Lisboa, Portugal, and 7Biomedical Sciences Institute Abel Salazar, University of Porto, Porto,
Portugal
Abstract
Forensic toxicology is the study and practice of the application of toxicology to the purposes of the law. The rel-
evance of any finding is determined, in the first instance, by the nature and integrity of the specimen(s) submitted
for analysis. This means that there are several specific challenges to select and collect specimens for ante-mortem
and post-mortem toxicology investigation. Post-mortem specimens may be numerous and can endow some spe-
cial difficulties compared to clinical specimens, namely those resulting from autolytic and putrefactive changes.
Storage stability is also an important issue to be considered during the pre-analytic phase, since its consideration
For personal use only.
should facilitate the assessment of sample quality and the analytical result obtained from that sample. The knowl-
edge on degradation mechanisms and methods to increase storage stability may enable the forensic toxicologist
to circumvent possible difficulties. Therefore, advantages and limitations of specimen preservation procedures are
thoroughfully discussed in this review. Presently, harmonized protocols for sampling in suspected intoxications
would have obvious utility. In the present article an overview is given on sampling procedures for routinely col-
lected specimens as well as on alternative specimens that may provide additional information on the route and
timing of exposure to a specific xenobiotic. Last, but not least, a discussion on possible bias that can influence the
interpretation of toxicological results is provided. This comprehensive review article is intented as a significant
help for forensic toxicologists to accomplish their frequently overwhelming mission.
Keywords: Ante-mortem and post-mortem forensic toxicology; biological specimens; collection procedures; stor-
age and preservation; interpretation.
Introduction to forensic toxicology courts rather than criminal courts. In any case, a good forensic
Forensic science represents the application of the differ- toxicologist must be prepared to answer several questions: The
ent scientific fields to the legal system proceedings. Forensic traditional one that must be answered is Has this person been
toxicology results from a hybridization of modern Analytical poisoned? Supplementary queries follow if the result is posi-
Chemistry and Fundamental Toxicology, and their applica- tive, such as What is the identity of the poison?, How was it
tion to the purposes of the law, in order to elucidate questions administered?, What are its effects?, and Was it a dangerous or
that occur in judicial proceedings related to intoxications. lethal amount? In other words, the forensic toxicologist should
Forensic Toxicology is concerned primarily with the medi- perform qualitative and quantitative analysis and must inter-
colegal aspects of the harmful effects of xenobiotics (XBs) on pret the probable role that a XB (or sometimes a endobiotic)
humans and animals (Langman and Kapur 2006). However, played in the case (Jickells and Negrusz 2008). According to the
the analysis and identification of medicines and the mainte- American Chemical Society, there are ~ 21 million registered
nance of agricultural, industrial, and public health legislation compounds, and the European Commission estimated that
(to ensure clean air, pure water, and safe food supplies) are also ~100,000 chemicals were in use (Greim and Snyder 2008)
fields of Forensic Toxicology, although associated with civil quite a daunting task for the forensic toxicologist.
Address for Correspondence: Ricardo Jorge Dinis-Oliveira, Institute of Legal Medicine, Faculty of Medicine, University of Porto, Jardim Carrilho Videira, 4050-167
Porto, Portugal. Tel: 00351 222073850. Fax: 00351 222083978. Email: ricardinis@sapo.pt & ricardinis@med.up.pt
(Received 09 April 2010; revised 12 May 2010; accepted 16 May 2010)
ISSN 1537-6516 print/ISSN 1537-6524 online 2010 Informa Healthcare USA, Inc.
DOI: 10.3109/15376516.2010.497976 http://www.informahealthcare.com/txm
364 R. J. Dinis-Oliveira etal.
In the field of Forensic Toxicology, poisoning (or intoxi- syndrome)) or own request (euthanasia), or by being the
cation) can be defined as: An individuals medical or social unwitting victim of intoxication, as in murder (attempted
unacceptable condition as a consequence of being under homicide), in sexual assault, or as consequence of
influence of XBs in a dose too high for the concerned person Mnchausens syndrome by proxy (the parent or guard-
(Uges 2001). For medical and legal purposes, it is important ian of a child exaggerates or creates symptoms of illness
to know how the victim became poisoned. All medications for their child/children in order to gain investigation,
can have unwanted but unavoidable side-effects. Doctors treatment, attention, sympathy, and comfort from medi-
must judge whether the cure is better than the ailment. cal personnel).
When a drug is deliberately or accidentally taken in such a
dose that a patients body cannot adequately metabolize and The field of Forensic Toxicology can be roughly divided
eliminate it, poisoning may ensue. A patient with terminal into three distinctly separate areas: Post-mortem Forensic
cancer and unbearable pain can receive strong painkillers, Toxicology and ante-mortem Human-Performance Forensic
such as morphine in high doses. The risk of such treatment Toxicology or Forensic XB testing (Goldberger and Polettini
is death of the patient, either directly or indirectly as a result 2002):
of the treatment. Is this acceptable? Do reasonable alterna-
tives exist? What would be the patients choices without a. In post-mortem Forensic Toxicology, the forensic toxi-
treatment? The inclusion of the term medical in the defi- cologist contributes to establish the cause and mode
nition of poisoning is important, as sometimes a doctor has of intoxication or death through the analysis of various
to exceed the maximum safe dose of a certain drug when a fluids and tissues obtained during autopsy. Some of the
disease is believed to be worse for the patient than the over- most notorious stories in the popular media are focused
dose (Uges 2001). By adding the clause social and/or legal to on death investigations;
the definition of poisoning, it is emphasized that the concept b. In ante-mortem Human-Performance Forensic
of intoxication depends on the victims normal state and on Toxicology, the forensic toxicologist is responsible for
prevailing cultural standards and laws. These factors strongly evaluating the role of XBs in the modification of human
differ from one country to the next (e.g. euthanasia, chemical behavior, usually applied to traffic safety and the respec-
abortion). tive operation of a motor vehicle, as well as doping in

Since poisoning is considered to be a medically or socially sport; and
unacceptable overdose of a XB, it is important to know how c. In ante-mortem Forensic XB testing, the forensic toxi-
somebody could be poisoned. In general, there are three pos- cologist is responsible for demonstrating prior use or
sible ways of causing poisoning (adapted from Uges 2001): abuse of selected XBs through the analysis of body flu-
a. Accidental poisoning, usually results from an accident, ids, usually urine. Results from these tests are usually
error, carelessness, or an unexpected situation in the applied to the workplace setting. People who work in
working environment. House accidents with babies law enforcement, many government agencies, and many
and children are also frequent due to their propensity to private companies are required to undergo drug testing
explore surroundings by putting things in their mouths. as a condition of their hiring or maintaining a job.
Intoxication due to medical or paramedical treatment,
Most cases that enter a Forensic Toxicology laboratory start
so-called iatrogenic intoxications, also belongs to the
with the suspicion that a XB is present in the body. The
category of accidental poisoning. Active therapeutic
identification and quantification of a XB is crucial and it is
drug monitoring can decrease the number of unwanted
highly dependent on continued improvements and on the
effects; however, if a doctor is charged with malpractice
development of new analytic instruments or techniques.
it is important to know whether it might be a case of
First, the forensic toxicologist will apply a screening test to
poisoning or just an unavoidable outcome. In the past,
establish whether there are any XBs present. Subsequently,
iatrogenic intoxication was accepted as a calculated
a qualitative or a quantitative confirmatory test is performed.
risk. Nowadays, patients are clients and thus less likely
Quantification of the XB is necessary to infer that the amount
to accept this risk, even resorting to civil or criminal
is compatible with fatal poisoning. Finally, interpretation is
proceedings to get compensation. Expert forensic toxi-
carried out. In laboratory medicine, the majority of labora-
cologists play a crucial role in such matters, weighing
tory errors result from or originate from the pre-analytic
the interests of the patient with those of the physician
phase and not from problems concerning the analytical proc-
or nurse objectively and honestly;
ess (Plebani and Carraro 1997; Witte etal. 1997). Therefore,
b. Experimental poisoning, commonly results from self- it must be recognized thateven with technological advanc-
medication or experimentation (even recreational) with esaccurate, forensically defensible results are predicated
drugs of abuse; and on the quality and type of specimens provided, and the
c. Intentional poisoning. Someone is intoxicated on documentation of each specimens origin and history. Thus,
purpose, either by self-administration (as occurs even an analytically accurate value may be subject to mis-
in attempted suicide or to get attention because of interpretation when the XB concentration in a single blood
psychological or social problems (himself Mnchausens specimen is used to explain the circumstances surrounding
Forensic toxicology 365
intoxication, particularly when the XB concentrations are not may be placed in a tamper-evident container labeled with the
excessively high or low. In principle, most of the pre-analytic case number and name initials. This protocol is particularly
pre-requisites for the quality of the samples considered in useful in institutions with large number of cases where speci-
clinical chemistry will also apply to the quality of ante-mortem mens may not immediately be transferred to the toxicology
and post-mortem specimens for Forensic Toxicology (Skopp laboratory. A unique identification number that accompanies
2004). However, toxicological investigation in ante-mortem the sample at all stages is a valuable safeguard (Moffat etal.
and post-mortem Forensic Toxicology must be regarded as 2004). Samples collected for clinical purposes (or even for the
unique. The pre-analytic phase covers a number of condi- coroner) are often not of evidential quality, but such samples
tions such as the request, the time interval between death may be all that is available. If appropriate, DNA testing may be
and finding of the victim, collection of biological material, its used to establish the origin of samples when there is concern
storage and transportation, registration, security, preparation over sample identity.
of the specimens including freezing, thawing, and aliquoting, Biological fluids can be collected using either wide-bore
administered therapies (Irjala and Gronroos 1998), as well as pipettes or disposable hypodermic syringes with appropri-
the involvement and the cooperation of various professionals ate needle gauges and lengths. For each specimen, separate
such as an emergency physician, the police, the mortician, disposable or clean devices and instruments such as scalpels,
and the autopsy staff, including the forensic toxicologist clips, and forceps should be used to avoid contamination.
(Skopp 2004). Additionally, samples must be collected in separate contain-
Up to date, best practice guidelines do not exist to assist ers, the collector must only be working with one specimen at
coroners and pathologists when toxicological analyses are a time, and the autopsy staff must maintain the cleanliness
required. In the following sections, an appraisal on pre-ana- of the specimen container (Karch 2008). All spillage on the
lytic aspects and procedures in ante-mortem and post-mortem outside of the container should be rinsed off and decontami-
toxicology based on current published literature and our own nated using 10% bleach solution (Karch 2008).
experience is given. The choice of sample container depends on its intended
purpose and should be decided by the analyst. Samples
Labeling, collection devices, and containers should be collected in such a way as to avoid both loss of
The first step in the specimen collection process (including analyte and the introduction of contaminating substances
evidence collection) is to ensure that the specimen contain- that could interfere with the analysis and interpretation of the
ers are labeled appropriately. It is essential that the sample analytical results. Usually, the best container to utilize when
and any intermediate containers used to carry it are labeled collecting and storing biological fluids or tissue specimens
in sufficient detail to eliminate any doubt about the sample is glass in appropriate storage racks, since glass is inert and
origin (Moffat et al. 2004). Without attention to this detail, does not contain any plasticizer contaminants (Moffat etal.
all other activities that occur with the specimen(s) may 2004; Jickells and Negrusz 2008; Karch 2008). Particularly, if
become compromised. As a minimum, the label should volatile solvents (e.g. resulting from solvent abuse or intoxi-
include the following information (Figure 1): (1) institutional cation with anesthetic gases) are to be analyzed, glass (with a
case number identifier or request number; (2) name of the cap lined with metal foil or Teflon-lined lids) is preferred (or
victim or other identifier; (3) specimen type (blood, liver, mandatory), in view of the fact that greater losses may occur
kidney, etc.) and anatomic place of blood collection, when if plastic containers are used. It is also important to collect
applicable (heart blood, femoral blood, etc.); (4) signature and seal the specimen in a container as soon as possible after
or initials of the collector; and (5) date and time of collec- opening the cadaver (Karch 2008). The tube should be as
tion. Finally, self-adhesive tamper-resistant stickers should full as possible to minimize headspace and should only be
be placed over both the specimen container and transport opened when required for analysis and only when cold (4C).
container lids to document specimen integrity (Karch 2008). With the exception referred for gases or volatile solvents,
The tape seal shall bear collectors initials and the collection plastic is almost always used, since it does not break, and
date. Alternatively, all collected specimens for a given case most types of plastic containers with screw caps are suitable
Institutional case number identifier or request

number:
Victim name:
Sample:
Blood collection:
Cardiac Peripheral Other
Date and collection time:

Signature:
Figure 1. Minimum recommendations for labeling containers adapted to the collection of routine forensic toxicological specimens.
for collection of fluid and tissue specimens. Whether a facil- time and expenses, by assisting the toxicologist to use the
ity chooses glass or plastic, it is important that the laboratory most useful methods of analysis and to interpret the results
carefully evaluates the potential contamination of the con- in the context of the case at a later stage. A copy of the post-
tainer before routinely collecting specimens in it, especially mortem report should be submitted as soon as it is available
when plastic is utilized. An example of the type of study that and a copy of any police sudden death report may also be
should be conducted is seen in that reported by Faynor and useful.
Robinson (1998). In addition, the nature and potential for Particular concern should be paid to the safety. As a
contamination can be evaluated by analyzing XB-negative general rule, samples should always be treated as if they are
biological fluids stored over time in the container. Methods infective and therefore must be handled with care, especially
using chromatography or mass spectrometry are particularly if originating from drug abusers. The major common risks
susceptible to some of these interferences as these materi- are associated with tuberculosis, hepatitis B, and human
als result in extraneous peaks, co-eluting peaks, and more immunodeficiency virus (HIV). Urine is the least likely to
subtle problems of ion suppression or enhancement (Zhang be infective. Staff in regular contact with potentially infec-
etal. 1996; Murthy 1997; Wu etal. 1997; Drake etal. 2004). tive materials must be properly trained in the safe handling
Neverthless, interferences from collection tubes extend and disposal of biological samples and should be vaccinated
beyond chromatography-based methods to other types of against hepatitis B, poliomyelitis, tuberculosis, and tetanus,
methods (Yen and Hsu 2004; Bowen etal. 2005). If plastic is and possibly other diseases in specific countries. Sample
used, it must be carefully chosen to ensure that it does not handling should be performed with due attention to prevent-
break when frozen (McCurdy 1987). For example, polysty- ing droplets splashing into the eyes and minimizing aerosol
rene is subject to cracking under these conditions, whereas formation (wearing eye protection and performing mixing
polypropylene is not. Plastic tubes containing separator and other procedures in a fume cupboard or microbiologi-
gels could constitute another source of interference (Dyne cal safety cabinet, using either sealable centrifuge tubes or a
etal. 1996; Dasgupta etal. 2000; Karppi etal. 2000; Marquet centrifuge with sealable rotors). Screw-capped sample tubes
etal. 2010). Once more, individual laboratories need to vali- are preferable to those with push-in stoppers as there is less
date the claim that these containers do not adsorb the XBs risk of aerosol formation when opening the tube (Flanagan
intented to analyze. The principles outlined by Bush etal. etal. 2007).

(2001) are a reasonable approach to such studies.
It is important that the container size chosen for each Specimens preservation and storage
specimen will allow it to be as close to full as possible in Reliable qualitative and quantitative toxicological analysis is
order to minimize concerns about oxidative losses due to the basis of a competent toxicological judgment and consul-
air trapped at the top of the container, volatile XB volatili- tation in Clinical and Forensic Toxicology. Unreliable results
zation, and salting-out effects from preservatives that may may lead to over-estimation or under-estimation of effects, to
be added to the tube (Moffat etal. 2004). Generally, 50-mL false interpretations, and to unwarranted conclusions (Peters
culture tubes represent the best choice for blood and urine etal. 2007). In the worst case, this might result in unjustified
specimens. Smaller tubes (e.g. 15, 20, and 30mL) can be used legal consequences or wrong treatment of the patient (Peters
for the collection of small amounts of blood, vitreous humor, 2007). Depending on the conditions and duration of handling
and bile specimens. In Figure 2 an example of suitable sample and storage, XB concentrations might have changed consid-
containers is given. It is recommended to put sample con- erably since specimen acquisition or since the time of death.
tainers inside another container for transporting. Specimens, especially if they cannot be analyzed immedi-
Finally, the correct collection of biological specimens ately, should be stored at an appropriate temperature, with an
for histological analysis is also important, since histology adequate preservative in case of demonstrated need, and in a
represents a very powerful tool in forensic analysis and for safe and suitable environment, only accessible to authorized
many times it has been used to corroborate other findings. In staff, to ensure security and integrity. With this procedure,
Forensic Toxicology, optical microscopy has been routinely it is possible to minimize interferences with the analytical
used. After collection, specimens are usually sectioned into methods and errors on the interpretation of the results. In
~ 5mm3 cubic pieces, fixed with 4% (v/v) buffered formal- some circumstances, these considerations need also to be
dehyde for 24hours and subjected to routine procedures for applied to the transport and continued storage of samples
paraffin histology (Pontes et al. 2008; Dinis-Oliveira et al. after analysis, sometimes for long periods, accordingly to
2009c). local judicial rules. This is because any unpredictable matters
may be disclosed by further investigation of the police.
Forensic toxicology request Until now, obligatory recommendations for specimen
An example of a Forensic Toxicology request designed to preservation and storage do not exist. Generally, once col-
accompany specimens to the laboratory is provided in lected, specimens should be stored in tightly sealed and well
Figure 3. Request forms should be filled in as complete as filled (but do not overfilled) containers at 4C for short-term
possible and submitted to the laboratory, together with the and 20C or preferably at 80C for long-term (more than
samples for analysis. The fields requested to fill up on this 1 week). Exceptions to this include hair and nail, which are
form are essential for an adequate analysis, and could save stable at room temperature, and filter-paper adsorbed dried
A D
B
C E
Figure 2. Sample and mailing containers. (a) Tube of 10mL capacity containing a fluoride salt (especially important for cocaine, carbon monoxide,
ethanol, hydroxybutyric acid, and cyanide) and ethylenediaminetetraacetic acid (EDTA, only in ante-mortem samples) for peripheral blood and the
mailing container. (b) Tube of 30mL capacity for cardiac blood, urine, and organs specimens, and the transport container. (c) Syringe of 2mL capacity
for gases analysis (namely in peripheral blood and vitreous humor) and mailing container. Air should be removed from syringe after sample collection
and analysis performed immediately. (d) Flasks adapted to histological analysis. (e) Security seals that can be used to complain with chain of custody.
blood, which is a convenient way of storing and transporting again after analysis. That will allow ethanol and other vola-
blood samples for specified analyzes if refrigerated transport tile solvents to evaporate, and therefore should be taken into
and storage is not feasible (Patchen etal. 1983; Croes etal. account if the sample is analyzed subsequently for forensic
1994; Howe and Handelsman 1997). Sealing is particularly purposes, sometimes several days later.
important in the interpretation of forensic results, especially Post-mortem specimens, more than any other specimen,
when volatile XBs are to be analyzed. Whole blood should not are likely to show some form of altered state because of either
be frozen, if plasma or serum is to be analyzed. For instance, an extended post-mortem period before collection or inad-
blood collected for clinical purposes is usually centrifuged equate or prolonged storage before analysis. Decomposition
to separate serum or plasma for testing on clinical analysers. and eventual liquefaction of tissues occur during post-mor-
Once separated, the serum or plasma vials may not be sealed tem periods and interfere with analytical results (Drummer
Using capital letter, fill the request in a
Forensic laboratory logotype

TOXICOLOGICAL complete and accurate way..
REQUEST Ante-mortem Post-mortem
Request number:_____________________________________________________________________________
Samples transport code:_______________________________________________________________________
IDENTIFICATION OF THE REQUESTING ENTITY
Name:______________________________________________ Telephone:______________________________
Officer/Investigation officer:______________________________ Telephone:______________________________
Address for report:_____________________________________ Judicial proceeding number:________________

VICTIM IDENTIFICATION
Name:_______________________________________________________ Birth date/Nationality:_____________
Male Female Weight:__________ Height:____________ Occupation (details of end-product of factory or

firm):____________________________________ Recent foreign travel:_________________________________
POLICE INVOLVEMENT
Is this case part of a criminal investigation or has it been submitted at the request of the coroner?
Yes No If yes, give details:_________________________________________________________________

___________________________________________________________________________________________
Are the analysis urgent? Yes No Telephone:____________

SUSPECTED ETIOLOGY
Driving accident Driver Pedestrian Passenger Homicide Suicide Home/leisure accident
Occupational accident Unknown Other:_______________________________________________________

Cause of death (if known):______________________________________________________________________
___________________________________________________________________________________________
TOXICOLOGICAL REQUEST REASON
Intoxication suspicion: Yes No If no, give brief details:___________________________________________

___________________________________________________________________________________________
Apparent natural death Death with traumatic lesions Drug abuse Rape Other:___________________
___________________________________________________________________________________________
REQUESTED ANALYSIS
Ethyl alcohol Carbon monoxide Drugs of abuse* Medicines* Pesticides* Other*

*Mention whenever it is possible , the XB(s) and/or EB(s) involved:_______________________________________
___________________________________________________________________________________________
Was any toxicological analyses performed before on either ante- or post-mortem samples?
Yes No If yes, give details of the results:______________________________________________________

___________________________________________________________________________________________
PAST OR RECENT MEDICAL HISTORY
Please detail any recent history of illness/disease. What drugs were prescribed?:__________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
Are the specimens likely to be infected with HIV, tuberculosis, hepatitis or any other serious disease?
Yes No If yes, give details:_________________________________________________________________
Figure 3. continued on next page

Figure 3. Continued.
INTOXICATION HISTORY
Time/Date of: intoxication________________________, onset of symptoms/signs_________________________,
hospital admission______________________________, when last seen alive:__________________, when body
found:________ Circumstances in which intoxication occurred: ____________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
Relevant symptoms: Diarrhea Vomiting Thirst Blindness Constipation Cyanosis Jaundice
Loss of weight Shivering Convulsions Miosis Mydriasis Delirium Coma Sweating

Affected organs , which:________________________ Others: Give details:__________________________

___________________________________________________________________________________________
Was any medical treatment or first aid given? Yes No If yes, please provide details on the drug/medication
given:______________________________________________________________________________________
given:
___________________________________________________________________________________________
Clinical features immediately before death (if applicable):______________________________________________
Time/Date of death (if applicable):________________________________________________________________
Where was the victim found (e.g. at work, in bed, outdoor):____________________________________________
Did he/she seem normal when last seen alive: Yes No If not, give brief details:________________________
___________________________________________________________________________________________
SAMPLES FOR ANALYSIS
Sample Number Time* Date* Quantity
Peripheral
Blood Cardiac
Other
Urine
Bile
Vitreous humor
Liver
Kidney
Organs Brain
Lung
Others
Gastric content (e.g. vomit and
gastric lavage or aspirate)
Others
*Time(s)/Date(s) of collection is particularly important for any samples taken prior to death, which are now being
submitted for analysis.
AUTOPTIC EXAMINATION
Collected data, namely odors, strange bodies, visceral congestion, skin, blood and mucosa color, putrefaction,
___________________________________________________________________________________________
___________________________________________________________________________________________
Medical doctor:_________________________________________________________ Telephone:____________
Time/Date:__________________ E-mail:________________________ Mobile phone:______________________
Place:____________________________________________________Signature:__________________________
A copy of any preliminary pathology report should be provided, if available. Report sent: Yes No
Figure 3. A complete toxicological request form covering all ante-mortem and post-mortem forensic investigations. XB, xenobiotic; EB, endobiotic.
2004). These phenomena are very much dependent on the amounts of -phenylethylamine are sometimes produced
interval between time of death and discovery of the body, by the action of bacterial translocation (Border etal. 1987).
the ambient temperature, and other environmental factors. On the other hand, fluoride preservation must not be used
One day in a tropical or very hot environment can induce when organophosphorous chemicals are involved. In sodium
significant putrefaction, while weeks at freezing temperatures fluoride preserved blood samples, dichlorvos is degraded
often results in little observable changes (Drummer 2004). completely within 15min (Moriya and Hashimoto 1999a;
The extent of these changes also varies significantly between Moriya etal. 1999).
XBs. The available data proves that the majority of XBs are sta- Esters, subject to alkaline hydrolysis, are more stable in
ble in blood, plasma, or serum samples under the conditions post-mortem blood than ante-mortem blood because the
usually encountered in a Clinical or Forensic Toxicology labo- pH of blood falls after death. Acidification may also stabi-
ratory. Instability usually occurs for XBs carrying ester moie- lize some labile conjugates such as N-glycosides or cocaine
ties, such as cocaine, heroin, or methylphenidate, containing (Moriya and Hashimoto 1996a).
sulfur atoms, such as phenothiazines or olanzapine, or other Ascorbic acid may be used as an antioxidant. For example,
easily oxidized or reduced structures such as nitrobenzodi- losses in olanzapine during storage at 4C may be reduced
azepines or dihydropyridine-type calcium channel blockers. by addition of 0.25% ascorbic acid (Olesen and Linnet 1998).
An excellent review on this topic was recently presented by However, the presence of an antioxidant may have reverse
Peters (2007). effects. During storage, the reduction has been observed of
Specimen preservatives are generally not required for the N-oxide metabolites of chlorpromazine in samples con-
specimens other than blood. Besides a preserved blood sam- taining antioxidants, artificially increasing the concentration
ple, an unpreserved specimen is also desirable (Skopp 2004). of the parent drug (Curry and Evans 1976).
Fluoride preservation with a final concentration of 15% An alternative way to preserve post-mortem fluids for
sodium (or potassium) fluoride by weight is usually recom- qualitative analysis is the use of filter paper (Skopp 2004).
mended. Only when a small amount of blood is collected, the This technique dates back to the early 1960s, when Dr Guthrie
excess fluoride may affect headspace volatile assays by alter- used dried blood spot specimens to measure phenylalanine
ing the vapor pressure of the analyte (Prouty and Anderson in newborns. Dried blood spot specimens represent a valu-
1987). Fluoride is mainly added to inhibit microorganism- able and cost-effective means for many clinical applications.
mediated conversion of glucose to ethanol, microorganism- In Forensic Toxicology, this technique has only tentatively
mediated oxidation of ethanol (Holmgren etal. 2004; Skopp been used in XB analysis (Chace et al. 2001). Up to now,
2004; Flanagan and Connally 2005; Jones 2006; Kugelberg and chloroquine, theophylline, and lead have been measured
Jones 2007; Bendroth etal. 2008), post-mortem conversion of from human blood collected and dried on filter paper (Mei
cocaine to ecgonine methyl ester by pseudocholinesterases et al. 2001). Analysis of blood spots dried on filter paper
(Isenschmid etal. 1989; 1992), enzymatic loss of other esters were shown to minimize the breakdown of cocaine and to
such as 6-acetylmorphine, and hydroxybutyric acid (GHB) largely preserve the profile of the parent XB and correspond-
production (Ferrara etal. 1993; 1995; Karch etal. 2001) after ing products of hydrolysis at the time of sampling. Due to
death and in stored samples. Therefore, when interpreting dehydration, both the chemical and enzymatic hydrolysis
forensic toxicological results, a serum or plasma specimen of cocaine, benzoylecgonine, and ecgonine methyl ester
collected in the emergency department might be expected could be stopped, and the analytical profile was ensured for
to contain higher concentrations of unchanged XB than a 17 days (Skopp 2004). The preservation of forensic urine XB
post-mortem blood sample collected later. However, this is specimens as dry stains has also been investigated. XBs such
often not the case, because post-mortem blood is regularly as amphetamine, benzoylecgonine, morphine, and phencyc-
collected in tubes containing fluoride, which avoids altera- lidine were stable under the storage conditions investigated.
tions of XB concentrations, whereas clinical samples are typi- An exception was 11-nor-9-carboxy-9-tetrahydrocannabinol
cally unpreserved. If high amounts of glucose and marked in urine spots, which degraded to 0 after 12 weeks of storage
contamination by bacteria are present, non-negligible at ambient temperature (DuBey and Caplan 1996). In clinical
amounts of ethanol can be produced in collected specimens. settings, sodium azide (NaN3) or sodium metabisulfite are
To discriminate ethanol produced post-mortem from the used as antimicrobial agents for urine samples. However,
ante-mortem one, n-propanol can be used as an indicator, production of hydroxybutyric acid occurred even in such
because it is concomitantly produced by bacteria. In that preserved samples stored at 4C or at a higher temperature
case, the concentration of n-propanol is not lower than 5% (Kerrigan 2002).
of a post-mortem ethanol concentration. The most typical Some benzodiazepines, particularly the nitrobenzodi-
amine produced during putrefaction is -phenylethylamine. azepines (nitrazepam, nimetazepam, flunitrazepam, and
Its structure is similar to that of amphetamines. The similar- clonazepam), are subject to post-mortem changes (Robertson
ity of the amine sometimes gives false positive results during and Drummer 1998). Nitrobenzodiazepines are actively
screening by immunoassays (Moriya and Hashimoto 1997b). converted to their respective metabolites by anaerobic
In analysis of XBs from specimens collected from polytrau- (enteric) bacteria in the post-mortem interval (Robertson and
matic victims, followed by intensive medical treatments, Drummer 1995; 1998). Consequently, flunitrazepam is rarely
special caution is needed. In such victims, non-negligible confirmed positive in blood specimens taken at autopsy. Post-
mortem specimens collected for nitrobenzodiazepines deter- ecgonine methyl ester (Moriya and Hashimoto 1996c; Logan
mination should be analyzed as soon as possible to minimize etal. 1997; Warner and Norman 2000). The pseudocholineste-
the loss of their respective 7-amino-metabolites. Noteworthy, rase activity in blood barely declines 3 weeks after its storage
the 7-amino-benzodiazepines are less stable than the par- at room temperature (Coe 1993). Ecgonine methyl ester is
ent drugs at 20C and require 60C for reasonable stability very rapidly further decomposed to ecgonine by chemical
(Robertson and Drummer 1998). Other benzodiazepines hydrolysis and thus does not accumulate in blood of living
are also subject to post-mortem changes, but these changes subjects (Moriya 2006). In the case of post-mortem blood,
can be minimized if specimens are stored at 20C or lower as mentioned above, the pH value rapidly declines due to
and analyzed promptly (El Mahjoub and Staub 2000). Data anaerobic glycolysis, resulting in the absence of chemical
suggests that many other benzodiazepines such as diazepam hydrolysis of cocaine into benzoylecgonine, but instead on
and temazepam are labile and are degraded under putrefy- the accumulation of ecgonine methyl ester through the action
ing conditions (Levine etal. 1983; Stevens 1984). This means of the coexisting cholinesterase. Therefore, the cocaine con-
that these drugs may not be detected at all in decomposed centration in blood at the point of death may be estimated
samples (Drummer 2004). When extensive putrefaction has by summing up the concentrations of cocaine and ecgonine
occurred and exposure to benzodiazepines is suspected, it is methyl ester (Isenschmid et al. 1992). While cocaine may
recommended to use other tissues where retention is more be stabilized to some extent (degradation still occurs) by
likely, such as hair (Drummer 2004). the addition of fluoride (a cholinesterase inhibitor)23
The stability of other drugs of abuse has been investigated weeks in a refrigeratorafter blood collection, the extent of
(Moody etal. 1992; Hippenstiel and Gerson 1994; Romberg breakdown between death and autopsy must be considered
and Past 1994; Giorgi and Meeker 1995; Levine etal. 1996; (Baselt 1983). Dugan etal. (1994) showed that cocaine con-
Golding Fraga etal. 1998). Heroin and cocaine are probably centrations in urine can change by as much as 37% over a
the most notable examples. Heroin and cocaine are not only 12-month period when stored at 20C, although other drugs
rapidly converted into their respective hydrolytic products in of abuse are reasonably stable. While some loss of cocaine
vivo, but also undergo rapid bioconversion in situ after death occurs in frozen specimens, it is not associated with forma-
(Drummer 2004). Moreover, unless special precautions are tion of ecgonine methyl ester (Levine etal. 1996).
undertaken, hydrolysis may even occur in the collection ves- Moderate losses for benzoylecgonine and 11-nor-
sel. Heroin is converted within minutes to morphine through 9-carboxy-9-tetrahydrocannabinol have also been
the 6-acetylmorphine intermediate. In fact, heroin is more reported in urine stored frozen (Romberg and Past 1994;
susceptible to decomposition by plasma cholinesterase than Levine et al. 1996). The concentration of the acid metabo-
cocaine, the half-life (t1/2) of the reaction in living subjects lite of 9-tetrahydrocannabinol, 11-nor-9-carboxy-9-
being only a few minutes. Therefore, it is difficult to detect tetrahydrocannabinol, shows significant decreases not only
heroin from the blood of a cadaver who had received intra- when urine is stored at room temperature for several days but
venous injection only several minutes before (Goldberger also after long-term frozen storage (Romberg and Past 1994;
et al. 1994); but 6-acetylmorphine, the main metabolite of Golding Fraga etal. 1998; Moody etal. 1999).
heroin, is relatively stable in blood and detectable post-mor-
tem (Goldberger etal. 1994). 6-acetylmorphine is relatively Chain of custody
labile in urine undergoing deacetylation to morphine at room One major difference between Forensic and Clinical
temperature, according to the pH of the specimen. However, Toxicology is that forensic work has legal repercussions.
6-acetylmorphine is stable in frozen urine (20C) for at least This implies that all evidence associated with a specific
12 months (less than a 2% loss) (Fuller and Anderson 1992). case must be kept in a secure area at all times and its
Morphine is relatively stable in specimens when stored frozen, lifetime must be documented by using a chain of custody
but shows significant losses when stored at 4C or higher for record (Karch 2008). This documentation is central to the
more than a few days, or in post-mortem specimens (Carroll demonstration that the evidence has remained intact, and
etal. 2000; Skopp etal. 2001). Of particular interest is the insta- not been adulterated, changed, mishandled, or misplaced
bility of morphine glucuronide conjugates. De-conjugation in any way that would compromise its integrity. Properly
of morphine metabolites to morphine has been observed maintained chain of custody documentation rules out any
in liver (Moriya and Hashimoto 1997a). This issue has been period of time in which a specimen may be left vulner-
recently discussed in the Shipman murders (Pounder 2003). able to adulteration or tampering (Karch 2008). Evidence
Of further interest is the variability in morphine and mor- ties together people, places, actions, and things that have
phine glucuronide ratios from different blood collection sites important impacts on circumstances surrounding events
(Skopp etal. 1996). These data suggest that morphine and in which individuals are held legally accountable. The
morphine glucuronide concentrations in the early stages of procedure also raises the issue of confidentiality and eth-
putrefaction, or when prolonged storage has occurred, may ics, where illegal activities may be involved (Wolff et al.
change substantially from the time of death. Cocaine is sub- 1999b). In criminal actions, the importance of the evidence
ject to chemical hydrolysis in the ante-mortem blood at pH7.4 may truly involve a life or death sentence, while in civil
to benzoylecgonine, and, to a minor extent, it is metabolized litigation large sums of money or property may be at stake.
by plasma cholinesterase (pseudocholinesterase) to yield Failure to properly document the chain of custody may
compromise not only the integrity of the specimen, but in cases of accidental poisoning, XBs to which the patient
also the credibility of the institution handling the speci- may have been exposed (Moffat etal. 2004). The informa-
men. Limited-access areas, at all times, with restricted tion exchange should be made between the doctor directly
access to only those individuals designated in the insti- treating the patient and staff of the Local Poison Information
tutions standard operating procedure, are mandatory. Center or any expert toxicologist for the specific XB. Their
The chain of custody data should include the dates and conversation, aimed to clarify symptoms, often yields clues
identification of individuals performing the sample col- for the cause of toxicity and for the priority of the analysis to
lection, transportation to the laboratory, and confirmation be performed. Close communication must continue if the
of receipt. Basically, the chain of custody should be able initial tests prove negative, so that the search can be wid-
to answer: who handled the evidence, what evidence was ened, or if the clinician requires advice on the interpretation
handled, when and why the evidence was handled, and of positive results (Moffat etal. 2004).
where the evidence was located at all times (Karch 2008). When the patients condition is severe and the diagnosis
Whenever the specimens are left unattended, they should is not clear, toxicological tests may be crucial. Analytical tox-
be secured in a locked container, refrigerator, or freezer. An icological results must be rapidly supplied (usually within
example of a chain of custody report is given in Figure 4. In 12hours of the patients arrival) without losing accuracy.
order to fulfill the chain of custody demands, samples and Preliminary screening tests may be required to narrow
toxicological request should be put inside an opaque plas- causative XB candidates before accurate analysis by instru-
tic bag sealed in such a way that any evidence of tampering ments of high performance. The identification of a causative
(tamper-proof containers) would be evident. XB is one of the most important tasks in emergency medi-
cine. Ideally, the XB can be both identified and quantified
within this time frame. When this is not possible, a qualita-
General aspects of ante-mortem tive result still has considerable value if the symptoms are
forensic toxicology consistent with the identified XB and should be given to the
When an intoxication incident occurs and the victim is sent clinician without delay (Moffat etal. 2004). However, they
to an health unit, medical doctors and co-medical staff must be of sufficient quality to allow an appropriate clinical
should concentrate their efforts on the patient intensive decision to be made. It is important to discuss the scope and
care (Suzuki and Watanabe 2005), irrespective of any other limitations of the tests performed with the clinician and to
aspects surrounding the case. Medical doctors and co- maintain high standards of laboratory practice, especially
medical staffs should communicate with the Local Poison when performing tests on an emergency basis. It may be
Information Center, giving as much information about the better to offer no result rather than misleading data based
patient as possible, namely clinical data (since databases on unreliable tests (Flanagan etal. 2007). Though clinicians
have been developed for estimation of a causative XB often treat poisoned patients on the basis of clinical history
according to clinical data), but also results of clinical tests, rather than wait for toxicological results, they may change
any previous medical history of poisoning, details of drugs their approach once they have them. In addition, the analy-
or other XBs to which the patient may have had access, and, sis should not end after the first positive finding because
Forensic laboratory logotype

CHAIN OF CUSTODY Using capital letter, fill the chain of custody
report in a complete and accurate way.
REPORT
VICTIM IDENTIFICATION
Name:_______________________________________________________ Birth date:______________________
Age:__________ Male Female
FROM TO
Name:_______________________________________
Confirmation of receipt:_________________________
Date/Time of collection transport :____________ Date /Time:___________________________________
N of the specimen handled: N of the specimen handled:

Why it was handled?___________________________ Why it was handled?___________________________
Name:_______________________________________
Confirmation of receipt:_________________________
Date/Time of collection transport :____________ Date /Time:___________________________________
N of the specimen handled: N of the specimen handled:

Why it was handled?___________________________ Why it was handled?___________________________
Figure 4. Example of a chain of custody report. Rows fromto should be repeted if necessary.
additional, hitherto unsuspected XBs may be present. Of Since the preliminary analysis may have been already car-
course, a positive result on a poison screening does not ried out, close co-operation between the forensic and hospi-
by itself confirm poisoning, because such a result may arise tal laboratories is desirable (Moffat etal. 2004). Frequently,
from incidental or occupational exposure to the XB in ques- victims of intoxication are admitted to hospital, even if they
tion or the use of drugs in treatment (Flanagan etal. 2007). survive only briefly, such as occurrences in traffic accidents or
Frequently routine clinical chemical tests will be per- overdoses. Even if death arises fairly soon after admission to
formed at one site, while more complex toxicological analy- hospital, it is common that blood, urine, and gastric content
sis will be performed by a different department, possibly at are collected as part of the medical evaluation and treatment.
a different location. Screening tests could be performed at Analysis of ante-mortem hospital admission specimens is
bedside or in the Clinical Laboratory, inside Health Care invaluable, for several reasons (Moffat etal. 2004), namely:
Unit, whereas complex qualitative and quantitative analysis
are usually performed at laboratories with more accurate a. Gives a good idea of the circulating blood concentration
analytical instruments and specialized analysts, such as of the putative XB at the time of admission to the hos-
local academic institutions or institutes of legal medicine. pital, which by definition is unaffected by post-mortem
However, the range of analyses that can be offered by spe- redistribution;
cialized laboratories to meet emergency demands usually b. May provide the only reliable indicator of dosage; and
encompasses only a few hundred XBs. Fortunately, in the
c. May provide the unique opportunity to perform mean-
vast majority of cases, the diagnosis can be made on cir-
ingful toxicology, if the person survives long enough
cumstantial and clinical evidence (Moffat etal. 2004).
for a XB to be eliminated from the body prior to death,
The analytical toxicological requests to institutions besides
or to be diminished to a concentration of limited or no
forensic ones is only possible when a poisoned patient is alive
forensic value.
and no criminality signs exist. Accidental self-poisoning and
attempted suicide cases are generally under the responsibility Even if blood or plasma collected on admission is not avail-
of the clinical toxicologist. Only a small proportion of these able, clearly timed specimens drawn several hours later may
cases should be referred to the forensic toxicologist if there still be useful if allowance is made for clearance and for the
are suspicions of criminality. Criminality cases include: presence of drugs administered as part of treatment.
a. Iatrogenic poisoning, in which a patient or relative sues a
health authority and its staff for neglection, because the General aspects of post-mortem
patient dies and a coroners inquest is ordered (Moffat forensic toxicology
etal. 2004);
Any violent, unnatural, sudden, or unexpected death should
b. Victims of so-called date-rape, who were administered be investigated to establish the cause and manner of death. In
XBs such as flunitrazepam or hydroxybutyric acid other words, any death that cannot be explained by a medi-
to induce confusion and amnesia and facilitate sexual cally recognized disease should be referred to the pathologist
abuse, and elderly abuse (Bechtel and Holstege 2007; for investigation, and Forensic Toxicology represents a strong
Kintz etal. 2008); ally. The aim of Forensic Toxicology is to help establish the
c. Non-accidental poisoning in children. Mothers are the role that XBs played in a death, or in events immediately
most frequent perpetrators of child poisoning and do so before death. It is the greater difficulty of interpreting post-
to attract sympathy and attention as a consequence of mortem results that most differentiates post-mortem Forensic
the childs illness (Mnchausens syndrome by proxy) Toxicology from ante-mortem Forensic Toxicology. In general,
(Meadow 1977; 1998; Bader and Kerzner 1999; Bappal a case falls under the jurisdiction of a legal medicine institute
etal. 2001; Aranibar and Cerda 2005; Carter etal. 2006; when the death (Stripp 2006):
Holstege and Dobmeier 2006). When these situations a. Results from violent, criminal, suicidal, or accidental
arise, the hospital toxicologist is obliged to take special means, including any death from criminal neglect or
precautions to conserve all residual samples and docu- due to suspicious or unexplained activity;
mentation that may feature subsequently as part of a
b. Occurs in an apparently healthy person with no
forensic investigation;
explained causes (indeterminate death);
d. Driving under the influence of alcohol or psychotropic c. Occurs during certain medical procedures;
substances;
d. Raises doubts if the victim is a fetal or a stillbirth case;
e. Issues relating to persons subject to disqualification by e. Occurs with no attending physician present;
their usual consumption of alcoholic beverages and
f. Occurs when the victim is incarcerated or confined; or
psychotropic substances; and
g. May have been caused by XBs poisoning.
f. Doping in sport and workplace drug testing
(Christophersen and Morland 1994; Botre 2003; Specimens are rarely ideal in post-mortem cases and without
Maravelias etal. 2005; Centini etal. 2007). specialist knowledge any results should be considered with
caution when attempting to interpret their significance. If or XB-related intoxications has not yet been established, but
the decedent was hospitalized prior to death, ante-mortem it is a broad consensus that collection should be performed
specimens, if available, should be submitted for analysis. In as early as possible after intoxication. It is also obvious that
addition, existing ante- or peri-mortem (at or near the time sampling procedure, selection of specimens, or quantity for
of death) specimens do not negate the need to collect post- toxicological analysis has to be case-dependent (Plebani and
mortem specimens. Carraro 1997), largely considering the case history, requests,
The development of gas chromatography (GC) and high- legal aspects, and availability.
performance liquid chromatography (HPLC) during the early Any specimen that was in contact with a XB is a potential
1970s had a major influence on the development and growth candidate for toxicological investigations, at least for qualita-
of pharmacokinetics and therapeutic drug monitoring (Karch tive analysis. Although toxicology analysis can theoretically
2008). As a result, the kinetics of XBs in clinical patients was be performed on almost any specimen, it is usually limited
easier to understand and predict. It was logical that toxicolo- to those for which there is an appropriate literature available
gists started to use the pharmacokinetic data obtained from to help in the interpretation of the results or for which there
living patients to interpret post-mortem blood concentrations, is a validated method (Moffat etal. 2004). Blood, urine, and
for example, to predict whether a given blood XB concentra- gastric content, namely gastric lavage fluid and vomitus, are
tion was in the therapeutic range, whether the blood level normally used as specimens for XB analysis in living sub-
was fatal, or even to predict the amount ingested prior to jects. In post-mortem toxicology investigations, available
death (Karch 2008). specimens can be numerous and variable (Skopp 2004).
Experience has since shown that post-mortem XB con- They may range from relatively pure solutions of a XB to
centrations must be interpreted from a very different per- a putrefying specimen. Proper collection and preservation
spective relative to those in living patients. Indeed, most XBs of post-mortem specimens is critical, since there is usually
measurements in samples from living patients are made on no opportunity to go back for re-collection of specimens, as
plasma or serum, and whole blood is used uncommonly occurs in cremation. Generally, the specimens routinely col-
(Ferner 2008). In addition, many processes (often refereed lected at autopsy include fluids such as peripheral and car-
as artifacts) occur after death that can change XB concen- diac blood, urine, bile, cerebrospinal fluid, vitreous humor,
trations, sometimes to a very large extent, which must be and gastric content and organs, particularly liver (Forrest
known in order to interpret results (Karch 2008). The pres- 1993). Kidney, brain, lung, spleen, and skeletal muscle are
ence of putrefactive changes of specimens limits the direct also occasionally collected for post-mortem studies. Blood
applicability of clinically validated assays in a post-mortem from peripheral sites should be obtained from femoral vein
setting. In addition, several alternative specimens can be and prior opening the thorax and abdominal cavities (Skopp
collected in a post-mortem setting. The period of enthusiasm 2004). Samples that may also be collected prior to organ
in the late 1970s and 1980s has given way to the realization dissection are urine, bile, cerebrospinal fluid, or fluid from
that there are many unique aspects of post-mortem toxicol- putrefactive blisters, hair, nails, and swabs from the orifices
ogy that must be considered when interpreting analytical of the body or from the skin (Skopp 2004). Organs are nor-
results (Karch 2008). It is no longer acceptable (indeed it mally collected after evisceration. They provide one of the
is impossible) to interpret post-mortem toxicology results best and most useful specimens to assist in the interpre-
from tables of so-called therapeutic, toxic, and fatal ranges, tation of blood findings. XB detection in tissue specimens
without taking into consideration the medical and case his- should be considered whenever highly lipophilic or prefer-
tory, presumed dose, autopsy findings, information from the ably bound to tissue XBs are suspected to be involved in
scene, the immediate circumstances of the death, the vari- the intoxication. Organs may also be useful in cases with
ous processes that can affect XBs concentrations both before extended post-mortem time periods, namely in decomposed
and after death, and the exclusion of other potential causes. bodies, and whenever body fluids are not available or dif-
For instance, a Swiss study has suggested that medical his- ficult to obtain. A large amount of data for XB findings in
tory plays an important role in interpreting post-mortem tissue exists, primarily for liver and kidney, and, to a lesser
data in ~ 70% of cases (Harding-Pink and Fryc 1991). It is degree, brain and lung (Baselt 2004). The analysis of tissue is
probably fair to say that many toxicologists and patholo- normally performed by weight, and therefore organ weights
gists are less confident about interpreting post-mortem XB must be recorded. Usually, 14g of tissue is homogenized
concentrations todaycomparatively to 1020 years ago. with four parts of water (or saline solution) to generate a
final dilution factor of 5.
Plasma, serum and blood are the ideal samples if quantita-
Collection of biological specimens for tive measurements are needed. Urine is commonly used in
toxicological analysis qualitative analysis since relatively large volumes are usually
In analytical toxicology, clinical chemistry and related fields, available (especially ante-mortem). The concentrations of
the words sample and specimen are used to denote a por- many XBs, and their metabolites, tend to be higher than in
tion of a body fluid, tissue, incubation medium, etc., collected blood, thereby facilitating detection. Gastric content can also
under defined conditions (Flanagan etal. 2007). Up to date, be useful specimens for identification of a XB when the time
a harmonized protocol for sampling in suspected poisoning after ingestion is short, since there are higher probabilities to
contain large amounts of an unchanged form of an ingested to ensure contact with the anticoagulant if one is being used.
XB. For the other specimens available, qualitative importance Even mild hemolysis will invalidate a serum iron or potas-
should also be considered, instead of quantitative analysis sium assay, and plasma or serum assays for other analytes
(Drummer 2004). concentrated in red cells such as chlortalidone (Fleuren
If an intoxication case is identified, additional questions and Van Rossum 1978; Guder 1986; Sonntag 1986; Delanghe
may arise such as the route of administration and an acute 2000). EDTA, citrate, or heparin are currently the used antico-
or long-term exposure to a XB. In several circumstances and agulants. Sodium citrate is normally used for clotting studies.
since there is a marked development of analytical technolo- Since tubes contain 0.5 or 1mL of the anticoagulant in aque-
gies, additional and alternative specimens such as hair, nails, ous solution, quantitative analysis is not suitable (Flanagan
sweat, or skin samples could be collected to complete the etal. 2007). Furthermore, dilution of the sample may reduce
toxicological investigation, because many XB are excreted the degree of plasma protein binding and consequently the
and accumulated into these compartments. If subcutane- plasma:red cell distribution of the analyte. It should also be
ous or intravenous XB application is suspected as the cause ensured that lithium heparin anticoagulant is not used if
of death, a sample of the particular skin region should be plasma lithium is to be measured (Flanagan etal. 2007).
excised together with a random specimen preferably taken If plasma or serum is required, these specimens should
from a similar (but different) site to act as a control (Skopp be separated from blood cells as soon as possible. Plasma
2004). A skin specimen or a cube of muscle taken from a sus- is obtained by centrifuging the tubes containing anticoagu-
pected injection site may support evidence of that route of lated whole blood at 20003000g for 10min and at 28C if
XB administration (Baselt 2004). Decomposed, skeletonized, necessary. If whole blood is allowed to stand (15min, room
or embalmed cases represent unique challenges for the toxi- temperature) in a tube without anticoagulant, a clot forms
cologist, due to the limited availability of specimens. In these that will retract sufficiently to allow serum to be collected
cases, muscular tissue, hair, and bone are normally collected (Flanagan etal. 2007). More plasma than serum can be sepa-
(Forrest 1993). In some cases, analyses of XBs in fly larvae, rated from whole blood.
in decomposing bodies, provides an insight to the presence To collect erythrocytes, heparinized blood should be
of XBs in the corpse. Soil samples collected at the site of a centrifuged (2000 g, 10min), the plasma, buffy coat, and top
skeleton or decomposed body, and even cremation ash are 10% of erythrocytes (mainly reticulocytes) removed, and
also possible specimens to be considered (Vass etal. 2008; the remaining erythrocytes carefully washed with isotonic,
Lombardi 2009). buffered saline to remove trapped plasma. The cells may be
Regarding the amount to be collect, there are no clear used directly or frozen, either to cause hemolysis, or for stor-
directives for sample sizes, but that should permit sufficient age. Platelets are usually isolated by the slow centrifugation
and sustainable conclusions to be made. Submission of very (e.g. 300 g, 15min) of anticoagulated whole blood to yield
small samples may result in reduced sensitivity and scope platelet-rich plasma, which is recentrifuged (2000 g, 10min)
of the analyses undertaken, but nevertheless such samples to harvest the platelets. Other white blood cells are most com-
should always be forwarded to the laboratory. General monly obtained by centrifugation through media of appropri-
requirements for sampling and the relative merits of each ate density (according to the manufacturers instructions) or
specimen are discussed in the following sections, and a isolated by solid-phase antibody techniques (Flanagan etal.
resume is provided in Table 1. 2007).
Arterial blood is normally collected by an experienced
Whole blood, plasma, and serum medical practitioner (it is a relatively dangerous procedure)
Ante-mortem blood specimens are generally collected from for the measurement of blood gases and is not usually used
the vein (usually the median cubital vein) using either a for toxicological analysis. Capillary blood, which closely
hypodermic needle and syringe (150mL) or a commercial approximates to arterial blood, can be obtained by pricking
vacuum-sampling system (Flanagan et al. 2007). A tourni- the heel, finger, or ear lobe; this procedure is most often per-
quet can be used to distend the vein prior to venepuncture, formed on small children.
but should be released immediately prior to sampling. Ante-mortem, most quantitative assays are carried out on
Disinfectant swabs that contain alcohols or iodine used to the plasma and/or serum (Chamberlain 1995), but antico-
clean the skin prior to venepuncture can contaminate blood agulated whole blood is essential if the XB is mainly associ-
samples and should not be used, especially when performing ated with the red cells (e.g. carbon monoxide, cyanide, lead,
ethanol analysis. In view of the requirement to prevent stress mercury). If necessary, whole blood can be stored at 20C or
on patients, 510mL of blood is enough. If the situation per- below, but freezing will lyse most cell types. Some XBs, such
mits, multiple samplings at different intervals are desirable. as many benzodiazepines, are extensively metabolized prior
For repeated sampling, a small cannula may be inserted into to excretion and then plasma is the specimen of choice for
a vein in the arm or hand, which allows venous access via a detecting the parent XB. Leaving plasma or serum in contact
rubber septum. If blood has been collected into a syringe, it with red cells can cause changes due to enzymatic activity
is essential that the syringe needle is removed and the blood or redistribution of an analyte between cells and plasma.
allowed to flow gently into the collection tube in order to Serum from coagulated blood can be used for the majority
prevent hemolysis. This should be followed by gentle mixing of the cases since the levels are almost always the same as
Table 1. Proposed guidelines to collect ante-mortem and/or post-mortem specimens with interest in Forensic Toxicology and specific comments about
advantages and limitations.
CARDIAC BLOOD
-30mL for plastic universal container with screw cap;
-Always collected;
-The label should detail the sampling site;
-Collection from right chamber is preferable;
-Normally used for screening (higher sample volume);
-Cumulative effect is possible due to post-mortem redistribution and diffusion, and putrefaction;
-Concentrations can be increased due to autolysis of cardiac tissue or due to trauma.
BLOOD CLOTS FROM SUBDURAL, SUBARACHNOID, AND/OR EPIDURAL SPACES
-Collected in traumatic cases;

-Are potential time capsules, since are generally poorly perfused, and therefore may reflect XB concentrations closer to the time of injury;
-Importance increases with survival time, especially if accurate injury time is known.
BLOOD FROM THORACIC OR ABDOMINAL CAVITIES
-Should be collected in traumatic cases;
-Only provides qualitative results.
EXHALED AIR
-Non-invasive;
-Collected for volatile XBs analysis;
-Non-invasive;
-Large volume available;
-Analytes must be volatile. Mainly used to assess ethanol ingestion and in carbon monoxide poisoning.
AMNIOTIC FLUID

-Useful to evaluate intrauterine XB exposure at an early stage of development;
-Minimal sample preparation;
-Easily applied to routinely used toxicology tests and relatively few interferences;
-Invasive sampling procedure that requires local anesthetic, ultrasound scan and highly trained medical personnel.
BILE
-All for a 10mL plastic universal container with screw cap (no preservative);
-Always collected;
-Collect prior to liver;
-Tie off gallbladder to reduce contamination;
-Important for XBs that exhibit enterohepatic circulation and chronic exposures;
-Often used for opioids;
-Analysis is difficult due to presence of bile salts;
-XBs concentrations may also be influenced by post-mortem diffusion from the liver and the stomach;
-Particularly useful when urine is absent.
PERIPHERAL BLOOD
-Always collected for complete toxicology analysis;
-Adult human body contains ~ 56 liters of blood;
-Identify the source and do not mix;
-Limited volume and more difficult to collect than cardiac blood;
-Post-mortem -venous femoral blood should be collected (vessel tied/clamped proximally near the inguinal ligament before sampling. The leg may be
slightly elevated to obtain more blood). Alternatively, venous subclavian or jugular blood;
-Ante-mortem usually blood is obtained from cephalic vein. Cord blood is obtained from the umbilical cord at parturition;
-Avoid contamination with cardiac blood;
-Preserve with a fluoride salt (such as sodium or potassium);
-If possible, reserve an aliquot without preservative;
-Tube must be filled completely to limit the headspace available, especially if volatiles are suspected;
-For analysis of gases or volatile XBs, a gas syringe should be used;
-Applied to the majority of quantitative analysis, although post-mortem interpretations are not straightforward;
-Low concentrations of basic and other XBs are normally present;
-Lithium heparin or EDTA should be used ante-mortem to obtain plasma;
Table 1. continued on next page

Table 1. Continued.
-Serum or plasma gel separator tubes, should be avoided, as some XBs may diffuse into the gel, leading to false low results;
-Plastic tube if paraquat suspected;
-If all blood is to be used, sample is mixed, and then frozen in order to rupture the cells before the analysis;
-Usually, venous cord blood is obtained when neonatal exposure is suspected. It may be possible to obtain plasma or serum depending on the volume
available. Accounts only for fetal XB exposure during the previous hours or days before collection and not for chronic exposure during the entire
gestation.
URINE
-30mL or all available for 30mL plastic universal container with screw cap (no preservative);
-Always collected;
-Submit any quantity, even if lower then 1mL, for immunoassay screening;
-Postmortem collection can be performed by inserting the needle directly above the pubic symphysis (no internal examination cases) or directly from
the bladder (internal examination cases);
-Is the ideal sample for screening approaches due to high concentrations of the parent XBs and metabolites;
-The bladder could be washed with a saline solution. If nothing was collected assure that vitreous humor is provided;
-No correlation exists with plasma level;
-Not useful for quantitative analysis;
-If death occurs soon after intoxication, results could be negative.
GASTRIC CONTENTS (INCLUDES VOMIT) OR STOMACH WALL IF GASTRIC CONTENTS ARE ABSENT
-30mL of the total homogenized for 30mL plastic universal container with screw cap (no preservative);
-30g of the stomach wall (no preservative);
-Usually collected for complete toxicology testing;
-Tie off stomach to prevent contamination of other viscera;
-Register all total volume in the labeler of the container;
-Only all amount is important and not the concentration. Large amounts can reveal overdose;
-Take care of poisonous gases if cyanides or phosphides have been ingested;
-Useful to guide blood analysis;

-Characteristic odors should be registered;
-Useful in sudden death due to oral poisoning since part of the XBs were probably not absorbed (tablets, capsules, etc);
-Medicines residues should be rapidly separated, dried and stored in a different container;
-Limited application in intoxications by intravenous and inhalation routes;
-Vomit and gastric aspirate are also important, especially the first sample.
CEREBROSPINAL FLUID
-All for a 10mL plastic universal container with screw cap (no preservative):
-Collected in advanced putrefied specimens;
-It is a plasma ultrafiltrate (i.e. similar composition to that of plasma except that high molecular weight proteins are absent) that surrounds the ele-
ments of the central nervous system;
-It is obtained by lumbar puncture (needle aspiration from the spinal cord);
-Rapid turnover time;
-XBs concentrations are generally higher in blood;
-No apparent correlation exists with blood XB concentrations;
-Only qualitative analysis.
BRAIN
-30g for a 30mL plastic universal container with screw cap (no preservative);
-Collected for lipophilic and volatile XBs analysis;
-Should always be submitted if the body is putrefied;
-Brain may be especially useful in infant XB deaths;
-Particularly useful in intoxications related to certain drugs of abuse (e.g. morphine, cocaine, etc) and other lipohilic XBs, namely organochlorated
insecticides;
-Can store volatile XBs;
-Can be the ideal matrix in advanced decomposition due to be distant from abdominal cavity;
-Concentrations may vary significantly from one region to another;
-Not expected to be affected by post-mortem diffusion and redistribution;
-The lipophilic characteristics cause analytic difficulties.
VITREOUS HUMOR
-All for a 10mL plastic universal container with screw cap (add preservative);
-Collected for ethanol and other drugs of abuse, and biochemistry analysis;
-Is the transparent, viscous fluid contained behind the lens in the eye;

Table 1. Continued.
-Is obtained by direct gentle aspiration from each eye using a 5-to 10-mL syringe and 20-gauge needle. The needle should be inserted through the
outer corner, until its tip is placed centrally in the globe;
-Low volume. Combine fluid from both eyes into a single tube;
-An appropriate amount of saline can be injected back into the eye in order to reproduce the cosmetic integrity of the eye;
-Only free XB is able to leave the blood and enter the vitreous humor;
-Lag behind blood levels ~ 1-2 hours;
-Valuable in the interpretation of post-mortem blood data;
-Could be very useful when blood is absent (e.g. trauma);
-It may be used to distinguish ante-mortem alcohol ingestion from post-mortem alcohol formation by fermentation;
-Less subject to contamination and putrefaction, and not affected by embalming and redistribution phenomena;
-Lacks the esterases that hydrolyze certain XBs and metabolites in blood and may be the specimen of choice to detect the metabolite of heroin,
6-monoacetylmorphine.
SPLEEN
-Collected for carbon monoxide and cyanide analysis;
-Very useful when blood not available in fire deaths and for XBs that accumulate in red blood cells;
-Quantitative results are not easily interpreted.
LUNG
-Colletced for volatile XBs and paraquat;
-Should be collected from the apex and in sealed container;
-Collect tracheal air as well;
-Quantitative results are not easily interpreted.
LIVER
-Always collected;
-Deep right lobe preferred to avoid contamination with diffusion of XBs from gastric contents;
-Identify source;
-Gall bladder should not be included with this sample;
-Useful for almost all XBs since it is the major metabolic organ and accumulates certain XBs (e.g. tricycle antidepressant).
KIDNEY
-Collected for metals or ethylene glycol analysis;
-Remove capsule;
-Could be important in absence of urine.
HEART
-Left ventricle;
-Not very useful except to help in the interpretation of blood data in digitalis intoxications.
HEAD HAIR FROM THE POSTERIOR VORTEX REGION OF THE SCALP OR THE BACK OF THE SKULL. ALTERNATIVELY, AXILLAR, PUBIC, ARMS
OR BEAR HAIR IF HEAD HAIR IT IS NOT AVAILABLE OR IF IT IS EXCESSIVELY BLEACHED OR PERMED
-Pen-sized bundle (150-200 hairs or 50mg);
-Collected for drug of abuse exposure history and heavy metals chronic exposure (As, Hg and Pb). More recently several drugs namely basic ones;
-Plucked in post-mortem or cut with scissor just near the root in ante-mortem cases;
-It is non-invasive and easy to perform;
-Sample not easily adulterated and in the case that there is a claim (sample switching, break in the chain of custody, etc.), it is possible to get an iden-
tical sample from the subject for re-testing;
-Store and align in aluminium foil. The proximal end should be identified (e.g. by tying with a peace of thread);
-Store at room temperature;
-The major practical advantage of hair testing compared with urine or blood testing is that it has a larger detection window (weeks to months, depend-
ing on the length of the hair shaft);
-It is not advisable to rely only on hair analysis, since it cannot provide adequate results, such as short-term information, for which blood and/or urine
are better specimens;
-Sample should be taken before the skull is opened;
-Usually available in advanced decomposition state and in exhumated cadavers;
-High sensitivity techniques are needed due to low amounts;
-The growth rate of hair is dependent to some extent on age, sex, anatomic region, race and health conditions;
-Segmentation is possible to access monthly exposure;

Table 1. Continued.
-The risk of external contamination leads to report false positive results;
-Not a suitable specimen for detecting recent XB use.
BONE
-Collected from skeletonised remains; Should be cut into small segments (e g femur rings) or crushed;
-Should be cut into small segments (e.g., femur rings) or crushed;
-There are no data to suggest that one anatomic region is better than another; however, large bones such as the femur are certainly easier to work with
than smaller bones;
-Only useful for qualitative analysis;
-Bone marrow may be useful when other samples are unavailable due to decomposition. It is encased in bone and it has a high degree of vascularity
and a lipid matrix that may act as a repository for lipophilic XB.
FLY LARVAE (MAGGOTS)
-Ten larvae randomly collected of an organ for a 30mL plastic universal container with screw cap (no preservative);
-Collected when decomposition prevents traditional specimens from being obtained;
-XB concentrations were found to depend on the tissue the larvae had fed as well as on their stage of development;
-Refer the organ of collection;
-Significant loss in XB concentration within 1 day after being removed from tissue containing XB;
-Qualitative analysis.
FINGER-AND TOE NAILS
-Whole nails should be lifted from the fingers and toes;
-Used to assess past exposure, particularly in newborns;
-Easy to store (room temperature);
-Provide a retrospective window of detection, even potentially longer than hair;
-Nail clippings from donors using Teflon-coated stainless steel scissors will be desirable to reduce contaminations;
-Considered discharged material and not influenced by melanin content.
FAT
-Collected for lipophilic XBs;
-Is not analyzed frequently due to the difficulty in reliably extracting XBs and because of substantial variability in parent compound/metabolite con-
centrations from one site to another;
-Collected from abdominal subcutis;
-Acts as a reservoir for many lipophilic substances;
-XBs identified in post-mortem adipose tissues reflect ante-mortem deposition and are not the result of post-mortem redistribution, diffusion, or
permeation.
SKELETAL MUSCLE
-Collected for most XBs;
-Normally collected from iliopsoas muscle (right or left of the lumbar portion of the spine);
-Can be useful in advanced decomposition since it is relatively less prone to autolysis;
-Data to aid interpretation is limited;
-Only important for qualitative analysis.
SWEAT
-Volume collected normally during one week;
-Useful for workplace drug testing;
-Can detect XBs up to weeks but inter-subject variability;
-Non-invasive;
-Not useful for quantitative work;
-Needs special collection devices and time to collect an adequate volume for analysis.
SALIVA/ORAL FLUID
-1-2mL for an appropriate collection container;
-Most XBs, namely drugs of abuse;
-Allows detection for hours or days;
-Relative noninvasive and observed collection. It is therefore resistant to adulteration and substitution;
-For some XBs, good correlation with free XB concentration in blood;
-Possible contamination with XBs taken orally;
-Requires sensitive immunoassays techniques and collection methods can dilute the specimen.
MECONIUM
-All available (2g minimum) for a 10mL plastic universal container;
Table 1. Continued.
-Always collected in suspected uterine exposure;
-Useful specimen to determine fetal XB exposure;
-XBs concentrations are generally higher than in urine because of accumulation over several months of gestation;
-Wide window for sample collection (20 weeks pre-partum);
-Viable analysis appears to be optimal via collection within 72h.
OTHER SAMPLES
-Faeces;
-Nasal swabs (fluid collected onto cotton swabs from inside the nose), peritoneal (fluid that accumulates in the peritoneum) and bronchoalveolar lav-
age (obtained by washing the bronchi/alveoli with an appropriate solution);
-Breast milk. The first expression of breast milk (colostrum, white to yellow pre-milk fluid) is especially rich in protein.
Notes:
-Ideally, ante-mortem specimens should be collected at the hospital entrance, before any treatments take place;
-Smaller volumes than those indicated may be acceptable (e.g. in the case of young children);
-Organs quantitative data is not easy to interpret but may be valuable in clarifying blood post-mortem data;
-Tissue specimens can also be obtained surgically or by biopsy in ante-mortem cases. Tissue obtained from an aborted fetus and/or placenta may sometimes
be presented for analysis.
those obtained from plasma, but some exceptions exist, as g. Range from 6090% in terms of water content;
it occurs in paraquat serum concentrations that are ~ 3-fold h. Be potentially dehydrated from exposure to heat during
lower than those in plasma obtained from the same blood a fire; or
sample (Dinis-Oliveira etal. 2008). For many analyses, serum
is preferred to plasma because it produces less precipitate (of i. Have a high degree of hemolysis, and for this reason
fibrin) on freezing and thawing and has the advantage that whole blood is normally analyzed directly.
there is no potential interfering additives. Its composition
is generally the same as plasma, except that fibrinogen and Ensuring that the body is stored at 24C prior to the autopsy,
factors associated with the clotting process are absent. and that it is processed as soon as possible after death, will
Post-mortem, whole blood is the specimen of choice for minimize the risk of changes in blood analyte concentrations
detecting, quantifying, and interpreting XB concentrations occurring before sampling.
since it is relatively easy to collect and most of the meaningful The general rules that the site or source of specimen col-
data derived from the literature was determined in serum, lection should always be clearly stated on the specimen con-
plasma and sometimes in whole blood (Leikin and Watson tainer and specimens taken from different sites should never
2003; Baselt 2008). Therefore, blood should always be col- be combined but always submitted separately, acquires more
lected. Nevertheless, it is important to take into account that post-mortem importance, since post-mortem redistribution
the specimen collected as blood at autopsy is not the same can cause the concentrations of many XB to vary markedly
collected in an ante-mortem venipuncture, and therefore clin- from site to site (Jones and Pounder 1987; Pounder and Jones
ically-based kinetic principles (mainly derived from serum 1990; Prouty and Anderson 1990; Pounder etal. 1996a). Blood,
and/or plasma) may not be directly applicable to post-mortem simply labeled as such, could come from almost anywhere
cases (Karch 2008). Indeed, blood obtained post-mortem is a for instance as pooled blood at the scene. Even XB concentra-
very variable sample. Normally it is considered to: tions in blood drawn from the same site, but simply placed
into different collection vials, can also sometimes differ by
a. Be relatively fluid but more viscous (may be clotted or several fold (Karch 2008).
completely fluid or partly clotted and partly fluid); Cardiac blood is usually more abundant than peripheral
blood, and XB levels in heart blood are generally higher than
b. Have typically numerous small clots (anticoagulants
in femoral venous blood. Therefore, post-mortem screening
are not recommended for post-mortem blood samples
toxicological tests may preferentially be performed on a heart
because these additives may affect XB concentration.
blood sample in opposition to urine, especially for XBs that
For instance, blood concentration of morphine in EDTA
are extensively metabolized, reserving the peripheral blood
tubes was 4.8% higher than in heparin tubes (Westerling
specimens for cases where additional context is needed for
etal. 1996));
interpretation. To obtain a proper cardiac specimen, when-
c. Possess sedimented cells; ever possible, the pericardial sack must be opened, the peri-
d. Be contaminated with tissue fluid before collection; cardium removed, the heart dried, and the blood specimen
removed using a syringe, preferably from the right chamber
e. Be potentially putrefied before collection; (Figure 5) (Karch 2008). It is essential to label the site from
f. Have a pH up to 5.5 (a sharp decrease in pH occurs which it was taken (right or left heart chamber) or to provide
immediately after death, which again slowly increases the information on mixed heart blood. Most toxicologists and
during the post-mortem interval due to the breakdown pathologists are well familiar with the widely discouraged
of protein); practice of drawing blood by a blind stick through the chest
A B
C D
Figure 5. To obtain cardiac blood, pericardial sack is opened and removed (ac) and a specimen is aspirated using a syringe, from the right chambe
(d).
wall (Karch 2008). Although such blood may be labeled as to the anatomical presence of a larger number of valves that
heart blood, it may contain pericardial fluid and/or pleural resist blood movement from the intestines (Harper and Couy
fluid. Since it could come from the pleural cavity, it might 1988). Therefore, this site is usually less, but not completely,
therefore be contaminated by gastric content, particularly affected by post-mortem changes in XB concentration, since
if the death was traumatic or decomposition severe (Logan redistribution from the bladder to femoral venous blood
and Lindholm 1996). Even blood drawn from the heart, after concentrations was observed (Moriya and Hashimoto 2001).
opening the body cavity at autopsy, may contain blood from Duplicate blood samples from distinct peripheral sites, e.g.
a number of sources, such as: the right and left femoral vein, may be taken to assure that XB
concentration had remained fairly constant post-mortem. The
a. Blood from one or more of the cardiac chambers; and peripheral blood specimen should be taken using a clean or
b. Blood that was drained from the pulmonary vein and new 1020-mL hypodermic syringe. The leg should not be
artery (and hence the lungs), from the inferior vena massaged in order to increase specimen volume. While it
cava (and hence from the liver), and from the aorta and is certainly a good practice to obtain, wherever achievable,
subclavian veins. peripheral blood, to avoid as much as possible the effects
of post-mortem redistribution or diffusion from the major
Accordingly, the so-called heart blood is potentially one of organs, interpreters should be cautioned that there is no such
the most non-homogeneous specimens in the body (Karch thing as pure femoral blood (Flanagan and Connally 2005).
2008). It is simply blood drawn from the site of the femoral vein.
Post-mortem blood for quantitative analyses is preferably Surely, if the proximal part of the femoral vein is clamped
obtained from the femoral vein prior to autopsy to avoid prior to sampling, it is likely that much of the blood will be
contamination with stomach contents and small pieces of peripheral and therefore relatively uncontaminated by
tissue(s) (Figure 6). Since many XBs are very potent, that is, blood from the major organs. Therefore, to obtain femoral
the blood concentrations associated to severe or fatal intoxi- blood, some forensic toxicologists advocate clamping the
cations are very low (typically mg/L or even g/L), even trace femoral vessels (Figure 6) (Yarema and Becker 2005). While
contamination of a peripheral blood sample can confound in many of the published studies on post-mortem redistribu-
the most careful analytical work. In such instances, toxico- tion the vessels have been carefully clamped prior to taking
logical analysis can often do little more than provide evidence blood samples, this is rarely done during routine medicolegal
of exposure to a particular XB (Flanagan and Connally 2005). autopsies, since this procedure results in added time to the
Leg veins are preferred to veins of the head and neck due autopsy examination as well as added incisions. Typically,
femoral blood is drawn by a stick to the unclamped femo- specimens where this may be useful (Jones and Pounder
ral vein in the groin area. In order to tie off the femoral vein, 1987; Pounder and Jones 1990; Prouty and Anderson 1990;
medical examiners must perform a cut-down incision over Pounder etal. 1996a). In some cases, e.g. in severe trauma, a
the proximal thigh to expose the vessel fully and the leg could peripheral specimen may be collected from the arm, specifi-
be slightly elevated to obtain more blood (Figure 6). If the cally from the subclavian vein.
volume drawn is relatively small (e.g. 25mL), it is unlikely Baselt (2008) and Leikin and Watson (2003) provided most
that much blood will be drawn from the central body cav- comprehensive data on a comparison of XB concentrations
ity, since it is also difficult to collect more than 510mL from in post-mortem cardiac and femoral venous blood. Many
a clamped femoral vein (Karch 2008). Without ligation, at XBs displayed a wide range of ratios of XB concentrations
least some blood will have been drawn down from the infe- in cardiac vs peripheral blood. Important factors affecting
rior vena cava, and hence from the liver, and from the larger the cardiac vs peripheral blood ratio are the type of XB, its
iliac vein. Notwithstanding these possibilities, Hargrove and volume of distribution, concentration range, protein binding,
McCutcheon (2008) showed good correlation between blind pKa-value, and the post-mortem interval between death and
stick femoral and clamped femoral samples for eight drugs autopsy. Generally, basic XB with a large volume of distribu-
from four different drug classes. These authors concluded tion showed the greatest range in cardiac vs peripheral blood
that a blind stick femoral blood sample does not have sig- ratios as well as the largest ratios.
nificant redistribution from central sites and is of equivalent
quality to a clamped femoral sample. An alternative sampling Blood clots and blood from thoracic and abdominal
technique is to cut the iliac vein at the side of the pelvis during cavities
autopsy, and to only sample blood that is massaged out from It is often common that a victim survives for several hours
the femoral vein directly into a test tube. Even if such a proce- after a fall or blunt trauma to the head, with circulation
dure ensures that the collected blood is from the femoral vein, remaining intact until the time of death (Kugelberg and
some post-mortem changes may just as well have happened Jones 2007). Owing to the reduced circulation in the dam-
in this blood too, e.g. diffusion from vessel walls and skeletal aged region of the brain, XBs in the blood clots (e.g. sub-
muscle. Since blood concentrations of some XBs may change dural, subarachnoid, and/or epidural) are not metabolized
markedly post-mortem, some authors advocate to analyze to the same extent as in blood circulating through the liver
blood obtained from more than one site, plus tissue or other (Kugelberg and Jones 2007). Accordingly, the blood clot
A B C
B
D E F
G H I
Figure 6. To obtain femoral blood, medical examiners must perform a cut-down incision over the proximal thigh (ac) to expose the femoral vein
fully (d, e) near the inguinal ligament, where it should be clamped (f ). The femoral vein blood can now be collected (g, h) and the leg could be slightly
elevated (i; arrows) to obtain more blood.
will contain a higher concentration of XBs compared with a Urine

specimen of peripheral venous blood obtained at autopsy. Urine represents one of the major routes to eliminate XBs
Therefore, the sampling and analysis of intracranial blood from the body. The accumulation of parent XBs and their
clots might furnish useful complementary information metabolites in urine usually results in high concentrations,
about the persons blood XB concentration several hours facilitating their detection. It is mostly used as a screening
before death, such as when the trauma/injury occurred. specimen (thought it is not always available), e.g. in death
As a result, blood clots are considered to be potential time related to drugs of abuse and prescribed medication as
capsules (Karch 2008). Nevertheless, it must be pondered well as in apparent accidental death where impairment is
that an injury often results in destruction of the skin sur- suspected (Drummer and Gerostamoulos 2002). The urine
face and surrounding tissue, which means that bacteria and matrix is generally devoid of circulating serum proteins,
infection can enter the wound, increasing the potential for lipids and other related large-molecular-weight compounds
microbial synthesis of ethanol in the blood clot after death. due to the glomerular filtration process. These characteristics
The usefulness of analysis in intracranial blood clots and greatly simplify the preparation of the specimen for toxico-
comparisons with concentrations in peripheral or heart logical analysis, enabling the investigation either directly
blood at autopsy has been reported before, especially for by immunoassays or non-instrumental spot tests as well as
ethanol (Moriya and Hashimoto 1996b; 1998b; Riggs etal. after extraction with an appropriate solvent (Skopp 2004).
1998; Takahashi et al. 1999; 2001; Kugelberg and Jones Detection times for XBs in urine can vary from 24h to as long
2007; Boonyoung etal. 2008). Blood clots may also be use- as a month, depending on the compound (Karch 2008). In
ful for documenting pre-existing XB use prior to hospital cases where death is suspected to have occurred rapidly due
therapy. to XB exposure, as might be suggested by the presence of a
Following severe injury or trauma, thoracic (pleural) and needle in the decedents arm at the time of death, negative
abdominal cavities blood should be collected for analy- urine findings are possible, and consistent if blood concen-
sis only if blood or uncontaminated blood clots cannot be trations of the analyte are very high. Thus, except for these
obtained from any other area. The composition of these acute XB deaths, where survival time is less than 1h (may not
blood specimens markedly differs from whole blood due yet have been excreted into the urine), urine provides an ideal
to the strong possibility of contamination by microorgan- matrix for the detection for the widest variety of XBs.
isms or with gastric and/or intestinal contents resulting It is almost universally accepted that, with few exceptions,
from severe trauma. For example, it is not uncommon that there is very little correlation between urine and blood XB
severe motor vehicle accidents result in rupture of the stom- concentrations, and even less correlation between urine XB
ach and diaphragm. If an autopsy is performed, the origin concentrations and pharmacological or toxicological effects.
and nature of the fluid so drawn should be obvious, and Therefore, quantitative measurements in urine are generally
hopefully noted (Karch 2008). However, if an autopsy is not of little use in toxicology (Suzuki and Watanabe 2005). To
performed and blood is sampled through the chest wall in interpret the context of exposure, blood should be tested for
an attempt to obtain cardiac blood, the coroner or medical the analytes found in the urine (Karch 2008). Indeed, many
examiner should be conscious that the sample is almost factors affect urine concentration, such as fluid intake, rate
certainly contaminated with gastric content. Trauma caus- of metabolism, glomerular clearance, urine pH (weakly
ing extended blood loss may also affect blood XB levels due basic compounds such as amphetamine or methadone are
to the physiological reactions, namely the increased heart more efficiently excreted in acidic urine, whereas weakly
rate and peripheral vasoconstriction, and plasma volume acidic compounds, such as barbiturates, are more efficiently
refill therapies. Hence, blood XB levels may increase or excreted in basic urine (Wolff etal. 1999a)) and the times of
drop, depending on their concentrations in the restora- voiding relative to the dose. Therefore, any attempt to pre-
tion fluid. Experimentally, codeine and morphine blood dict or even estimate a blood concentration from a urine
levels were found to increase significantly after controlled concentration is pure imprudence. The wide variation in its
exsanguination in rats (Kugelberg et al. 2003; Jones et al. composition can be corrected by the creatinine value of the
2008) and a similar study showed that the analgesic effect particular sample (Skopp 2004).
of morphine incresed when given to rats with hemorrhagic A volume of 30mL or all available urine (no preservative)
shock (De Paepe etal. 1998). Although further studies are is sufficient for most purposes and samplings obtained at
needed to determine the influence of such conditions on time intervals are preferable. When urine is obtained by cath-
ante-mortem redistribution for several XBs with different eterization from a patient, it should be considered the pos-
pharmacokinetic properties, the phenomenon should be sibility of being contaminated with a local anesthetic that was
considered in trauma cases with longer duration of blood applied to the catheter as a gel formulation. During autopsy,
loss. Nevertheless, qualitative documentation of the pres- urine specimens should be taken by insertion of a clean/
ence of given analytes is of importance and value in death new hypodermic needle into the bladder (Figure 7) (Karch
investigations with respect to compliance and exposure 2008). For victims not subjected to internal examination, the
issues (Karch 2008). Therefore, these blood-like samples needle may be inserted directly through the lower abdomi-
only provide a qualitative documentation of the presence nal wall, just above the pubic symphysis (Figure 7) (DiMaio
of a XB. and DiMaio 2001). In cases where the bladder appears to
A A
B
B
Figure 7. To obtain urine, for victims not subjected to internal examina-

tion, the needle may be inserted directly through the lower abdominal
wall, just above the pubic symphysis (a). For victims subjected to internal
examination, urine specimens should be taken directly from the bladder
(b).
C
be empty, it is important to aspirate as much urine as pos-

sible from the bladder and the ureter (Karch 2008). Bladder
washing using a minimum amount of clean water (or saline)
is desirable in the absence of any urine. The specimen con-
tainer should clearly identify and indicate the nature of this
specimen, and the amount of water/saline utilized.
Bile
Bile is the thick yellow-green fluid secreted by the liver via the
gall bladder into the intestine. It represents a collection and
storage depot for many XBs and corresponding metabolites,
Figure 8. Bile is collected prior to liver. Gallbladder is exposed (a), should
namely those that undergo biliary excretion by being sub- be tied to reduce contamination, and bile is aspirated using a clean/new
strates of P-glycoprotein efflux transporter and are often sub- hypodermic syringe (b, c).
ject of enterohepatic circulation (Elferink etal. 1995; Fardel
etal. 2001; Dinis-Oliveira etal. 2006b; 2008). It is a useful fluid
for qualitative analysis and can be used in screening when corresponding blood specimens (Vanbinst etal. 2002), the use
urine is not available. Biliary XBs concentrations may also of a smaller sample volume and dilution into a buffer is rec-
be influenced by post-mortem diffusion from the liver and ommended, together with a clean-up step. Cocaine and major
the stomach (Karch 2008). Bile is aspirated from the gallblad- metabolites such as ecgonine methyl ester, benzoylecgonine,
der using a clean/new hypodermic syringe (Figure 8). Due to and cocaethylene were shown to be present in bile in levels
its complexity (it contains high concentrations of bile acids 36-times higher than in blood (Agarwal and Lemos 1996).
and other endogenous substances), methods developed for In several cases, although the XB was not detectable in blood,
other fluids with well-established consistency may not be it could be identified in bile (Vanbinst etal. 2002). For these
immediately adaptable to bile (Agarwal and Lemos 1996). reasons, there is a much smaller probability for a XB to go
Because XBs concentrations are often higher in bile than undetected if bile is analyzed in addition to blood samples.
Bile had been used in cases of chronic heavy metal poison- fact that it is often more difficult to collect than blood post-
ing or when XBs such as morphine, chlorpromazine, or col- mortem. In spite of these limitations, Engelhart and Jenkins
chicine had been implicated (Drummer and Gerostamoulos (2007), in an excellent experimental work and revision of
2002). A summary of the distribution of benzodiazepines, literature, stated some conclusions:
antidepressants, cocaine, and some miscellaneous XBs in
bile, blood, and liver specimens was previously provided by a. XBs are detectable in cerebrospinal fluid;
Agarwal and Lemos (1996) and Vanbinst etal. (2002). b. Concentrations measured are within the analytical capa-
bility of current testing technologies;
Cerebrospinal fluid and vitreous humor c. XBs concentrations are generally higher in blood than
Cerebrospinal fluid and vitreous humor are aqueous saline in cerebrospinal fluid;
solutions, transparent, clear, and free of clotted material. They
d. No apparent correlation exists between blood and cer-
are less subject to contamination and bacterial invasion by
ebrospinal fluid for XB concentrations;

virtue of their protected environment inside the brain, the
spinal column, or the eyes. Cerebrospinal fluid is thought e. Cerebrospinal fluid XB concentrations should not be
to be closer to the site of action of several XBs than blood, used to estimate blood concentrations; and
and are useful for screening analysis (Maurer 1999). Both f. Cerebrospinal fluid /blood ratio should not be used in
cerebrospinal fluid and vitreous humor also contain very lit- isolation to differentiate XB intoxication from an inci-
tle enzymes and proteins. Therefore, XBs, which are highly dental finding.
protein bound or those that are lipophilic, tend to be found
in lower concentrations in these fluids than in blood (Forrest Vitreous humor
1993). The vitreous humor is located between the lens and the retina
and fills the center of the eye. The vitreous humor is filled
Cerebrospinal fluid with a transparent, delicate connective tissue gel called the
Cerebrospinal fluid is formed by the choroid plexus, a gel vitreous or a transparent liquid called the liquid vitre-
specialized tissue located within ventricular cavities of ous (Jenkins 2008). The gel vitreous is a collagen gel that is
the brain (Redzic et al. 2005). Cerebrospinal fluid has a water-insoluble and liquefies with age such that the adult eye
total volume of ~ 100160mL and is produced at a rate of contains only liquid vitreous. In Forensic Toxicology, the gel
2540mL/h, resulting in a turnover time of 36h. The pro- vitreous and the liquid vitreous are considered one specimen,
tein content of cerebrospinal fluid is low, and estimated to the vitreous humor. The vitreous humor consists mainly of
be 0.30.6% that of plasma. XBs may gain entry to the cer- 99% water (as a result, the specimen is easy to work), col-
ebrospinal fluid directly through the choroid plexus, which lagen is the major structural protein and hyaluronic acid is
acts as a bloodcerebrospinal fluid barrier. XBs may also the mucopolysaccharide (Jenkins 2008).
enter the cerebrospinal fluid indirectly by passage across Vitreous humor is obtained by direct gentle aspiration from
the bloodbrain barrier, followed by transport from the each eye using a 510-mL syringe and a 20-gauge needle.
interstitial fluid to the cerebrospinal fluid (Shen etal. 2004). The needle should be inserted through the outer corner (just
Suboccipital puncture is favored to collect cerebrospinal above the junction between the upper and lower eyelids),
fluid post-mortem. Alternatively, it may be aspirated from until its tip is placed centrally in the globe (Figure9). Vacuum
the ventricles after the skull has been removed. Normal tubes and heavy suction should be avoided to prevent speci-
cerebrospinal fluid should be clear, colorless, and free of men contamination with retinal fragments and other tissues
clotted material. (Karch 2008). It constitutes 80% of the eye and with proper
The scarce database of reference values that exists for cer- techniques, 23mL of fluid can be removed from each eye
ebrospinal fluid restricts its applicability for the interpretation in an adult, while up to ~ 1mL of specimen may be removed
of the analytical findings. Other drawbacks lie mainly in the from a newborn (Coe 1993). Once the vitreous specimen has
Figure 9. Vitreous humor is obtained by direct gentle aspiration from each eye using a 510- mL syringe. The needle should be inserted through the
outer corner (5mm lateral to the limbus (corneo-scleral junction)), until its tip is placed centrally in the globe.
been collected from the eye, an appropriate amount of saline Wyman and Bultman 2004). This justifies why 6-acetyl-
can be injected back into the eye in order to reproduce the morphine might be present in vitreous humor, indicating
cosmetic integrity of the eye. Fluoride preservation is recom- heroin consumption, although blood results are negative
mended for this specimen, particularly for ethanol quantifi- (Pragst etal. 1999a; Scott and Oliver 1999a);
cation in diabetes-related deaths (Skopp 2004). Specimens d. Because the eye is remote from the central body cavity
obtained from both eyes are usually combined in one prop- and the abdominal organs, it has been suggested that
erly labelled specimen container, but different opinions on vitreous humor may be a useful fluid for the determina-
this procedure exist (Karch 2008). tion of XBs that are subject to post-mortem redistribu-
Interpretation of vitreous XB concentrations is difficult for tion. Although that may hold true for many XBs such
several reasons: as digoxin, it has also been shown for others, notably
cocaine and 3,4-methylenedioxymethamphetamine,
a. Very few studies have been published that relate blood
an increase of the concentration in the vitreous humor
concentrations to those in vitreous humor;

after death. The authors suggested that XBs stored in
b. Large ad hoc data on vitreous XB concentrations is frag- the globe wall or brain had been released into vitreous
mented in innumerable case reports (De Letter et al. humor (McKinney etal. 1995; De Letter etal. 2000);
2000; 2002; Osuna et al. 2000; Scott and Oliver 2001;
e. By virtue of its avascular and cellular nature, and protected
Hardin 2002; Elliott 2004; Skopp 2004; Teixeira et al.
environment inside the eye, it is less subject to contamina-
2004; Duer etal. 2006); and
tion and early bacterial decomposition that typically occur
c. Equilibrium between the blood and vitreous is slower in blood, charring, and trauma, and therefore it may be
than among the blood and other extracellular fluids. In used to distinguish ante-mortem alcohol ingestion from
vitreous humor, a delay in the uptake of XBs is likely to post-mortem alcohol formation by fermentation and may
occur, and, inversely, there seems to be a delay in the provide the only opportunity to establish an ante-mortem
excretion process. This suggests the presence of a barrier ethanol concentration in embalmed bodies (Caplan and
that is called the bloodvitreous barrier (Jenkins 2008). It Levine 1990; ONeal and Poklis 1996). Nevertheless, some
has been observed that vitreous XB concentrations often
studies comparing vitreous humor and blood ethanol

reflect XB blood concentrations 12hours prior to death concentrations yielded a wide variety of ratios of vitreous
and that any XB found in the blood will be detected in humor to blood ethanol (Caplan and Levine 1990). If such
the corresponding vitreous humor specimen, given ana- diversity is seen for an analyte that demonstrates mini-
lytical techniques of sufficient sensitivity (DiMaio and mal post-mortem redistribution effects, attempting to use
DiMaio 2001). vitreous humor XB concentrations to aid in interpreting
heart blood XB concentrations may prove difficult (Prouty
In spite of these limitations there are some advantages of and Anderson 1990); and
humor vitreous specimen to be considered:
f. It has been shown to be particularly useful for the post-
a. A large number of XBs, including barbiturates, metha- mortem analysis of glucose, urea nitrogen, uric acid,
nol, cocaine, morphine, tricyclic antidepressants, creatinine, sodium, and chloride. Measuring these ana-
digitalis-glycosides, benzoylecgonine, acetaminophen, lytes is important for documenting diabetes, degree of
salicylate, and benzodiazepines have been analyzed hydration, electrolyte balance, the post-mortem interval,
(Engelhart and Jenkins 2001; Hardin 2002; Elliott 2004; and the state of renal function prior to death (Coe 1993;
Teixeira etal. 2004; Parker and McIntyre 2005; Favretto James etal. 1997; Osuna etal. 1999; 2001; 2005; Tagliaro
etal. 2007; Pontes etal. 2009); etal. 2001). Vitreous humor concentrations of sodium
and chloride approximate the serum concentrations of
b. XBs that tend to be somewhat hydrophilic at physi-
these ions in healthy adults, especially in the early post-
ological pH are more likely to have concentrations
mortem period. Potassium concentrations in the vitreous
approaching those in blood or plasma than those XBs
humor increase rapidly after death as potassium leaves
that are either highly protein bound (e.g. tricyclic anti-
the cells into nearby fluids (Prasad etal. 2003). Vitreous
depressants) or highly lipophilic (e.g. benzodiazepines).
humor calcium concentrations are comparable to serum
In fact, a significant negative correlation between the
calcium concentrations. Two nitrogenous compounds,
vitreous:blood concentration ratio and the degree of
urea and creatinine, are also present in concentrations
protein binding of different XBs has been reported,
similar to serum and are stable during the early post-
since XBs move in and out of the vitreous by diffusion
mortem period. In post-mortem cases, these endobiotics
and therefore only free XBs are able to leave the blood
are routinely measured as indicators for the serum con-
and enter the vitreous (Holmgren etal. 2004);
centrations of these substances at death (Coe 1993).
c. Lacks the esterases that in blood (in vivo and in vitro)
hydrolyze the metabolite of heroin, 6-acetylmorphine to Brain
morphine (Bogusz etal. 1997a; b; Guillot etal. 1997; Lin XB concentration data determined in brain tissue are not
etal. 1997; Pragst etal. 1999b; Scott and Oliver 1999b; hard to find in literature (Moriya and Hashimoto 1996c; 2003;
Kalasinsky etal. 2000; 2001; De Letter etal. 2002). The brain aliquoting. If this is not possible, then the total volume
is a useful specimen for the measurement of XBs because it present must be noted and provided with a representative
is the primary site of action for many lipophilic XBs (Skopp aliquot (~ 30mL) to the laboratory to allow the calculation
2004). It is also a very valuable specimen for the measure- of the total amount of the analyte present in the stomach.
ment and interpretation of XBs since it is remote from the The total amount of a XB remaining in the gastric content
stomach and other major organs in the body and therefore it is more important than its concentration. An estimation of
is expected not to be affected by post-mortem diffusion and the amount of XB present in the gastric volume is helpful to
redistribution. In addition, because the brain is in a protected decide whether an analytical finding is rather more consist-
environment, it also tends to be more resistant to post-mor- ent with an overdose or a therapeutic dosage taken just prior
tem decomposition, comparatively to other tissues or blood to death (Skopp 2004).
(Sawyer and Forney 1988). Also, metabolic activity is lower in Gastric content can replace urine in toxicological screen-
the brain than in other tissues or blood. For example, chlo- ing, namely for those XBs that are difficult to detect in blood
roform undergoes considerable biotransformation in man, or urine. However, as with urine, quantitative analyses serve
and less than 0.01% of a dose can be found in urine (Fry etal. no purpose, since gastric levels do not reflect the amount of
1972), though high concentrations were observed in brain XB absorbed (Jickells and Negrusz 2008). Moreover, gastric
specimens from acute fatalities (Gettler and Blume 1931). content is useful to determine the time since XB intoxication
Moriya and Hashimoto (1996c) demonstrated that cocaine and to distinguish oral from other routes of administration.
was stable in decomposed homogenates of human brain at A large quantity of the parent XB in the gastric content, rela-
2025C and at 37C over an observed period of 24h. In rab- tive to a prescription dose, is indicative of an oral overdose
bit brain specimens, cocaine degraded much more slowly when supported by blood and/or tissue findings (Wetli and
than in blood or liver over a period of 5 days (Moriya and Mittlemann 1981; Bar-Or and Wahby 1982; McCarron and
Hashimoto 1996c). These factors increase the likelihood of XB Wood 1983; Joynt and Mikhael 1985; Hassanian-Moghaddam
detection in brain specimens compared with other tissues, and Abolmasoumi 2007; Schaper etal. 2007; Takekawa etal.
especially for XBs with low stability (e.g. cocaine). However, 2007; Sengupta and Page 2008). Nevertheless, a low absolute
in most case reports, it was not specified which anatomic amount present in the stomach content does not rule out the
region of the brain tissue was collected for analysis, and cur- possibility of an overdose (Skopp 2004). Numerous cases have
rent data establish that concentrations may vary significantly shown that it may take several hours for an individual to die
from one region to another. In an olanzapine-related death, from an intentional overdose, depending on the XB ingested,
concentrations ranged from 0.17 in the left frontal cortex to the amounts, co-ingestion of alcohol, general state of health,
0.86mg/Kg in the midbrain (Merrick etal. 2001; Horak and and age. It is not unusual for victims to die from an oral over-
Jenkins 2005). Therefore, the brain cannot be regarded as a dose with less than a single therapeutic dose remaining in
single pharmacokinetic compartment, which is in accord- the stomach, notwithstanding the fact that an overdose of
ance to its complex structure (e.g. lipid contents are higher XB can irritate the stomach lining and therefore delay gastric
in white matter than in gray matter). Until now no guidance emptying. Extensive vomiting before death can also reduce
exists stating that one part should be collected over another. the amount of XB remaining in the stomach at the time death
Thus, the area of the brain from which the specimen is taken (Karch 2008). In addition, the toxicologist is cautioned that low
must be carefully documented. gastric content of a XB does not necessarily mean that the XB
was recently consumed, or even prove that the XB was taken
Gastric contents: Vomitus, gastric aspirate, orally. This may represent passive diffusion (re-excretion into
and lavage fluid the gastric contents through gastric fluid) and/or ion trapping
Ingestion is the preferred route of intoxication (Klaassen from the blood back into the stomach contents via the gastric
2008). Oral overdoses, whether by accident or by intent, may juice, a phenomenon frequently observed, especially in XBs
be readily discovered through the analysis of gastric content and its metabolites that are weakly basic in nature. The same
(i.e. vomitus, gastric aspirate, and lavage fluid, or obtained re-excretion phenomenon can be seen with XB metabolites
during autopsy), especially if information on the XB used is where, invariably, concentrations can be found in the gastric
not available and when specimens are obtained soon after content. In addition, small quantities of XB can derive from
the intoxication. bile, especially during agonal processes when vomiting of bile
Sampling of gastric content is usually performed dur- can occur (Karch 2008). Conversely, the presence of ghost
ing autopsy. The pathologist should tie off the stomach tablets in gastric content has been reported for at least one
ends before removing it in block from the abdominal cavity type of slow-release analgesic, where overdose or abuse was
(Figure10). The stomach should be opened away from other not suspected. Apparently, the wax-resin matrix of these sus-
specimens and tissues to avoid contamination of other vis- tained release tablets may remain in the gastric content long
cera (Karch 2008). Because gastric content is not homogene- after the XB has diffused out (Anderson etal. 2002).
ous, and since the total volume of gastric fluid is important Remains of undegraded tablets, capsules, or other materi-
for the interpretation of positive findings, the entire contents als of exogenous evidence, usually present if specimens are
of the stomach, without preservative, should be collected collected soon after intoxication, should be transferred to
and submitted to the toxicology laboratory for mixing before a separate container. Large amounts of capsules or tablets
A B
C D
E
Figure 10. To collect gastric contents, the pathologist should tie off the stomach ends (ac) before removing it from the organ block (d). The entire con-
tents of the stomach should be collected and mixed before aliquoting (e).
may form a gelatinous mass, which is not readily dissolved or capsules by weight, markings, colour, shape, and possibly
broken up, and therefore can be found in the stomach many other physical characteristics.
hours, or even a day or two, after an overdose; they are called Pesticides or solvents may readily be discovered due to
bezoars (Ku 1996). They occur, at least in part, because gastric their peculiar odor or conspicuous appearance. The odor
emptying time is delayed significantly by irritants, including of gastric content can potentially point to a specific XB that
large amounts of undisolved XBs residues. However, the might otherwise elude routine detection in the toxicology lab-
phenomenon is also occasionally seen in patients where oratory. Cyanide ingestions produce stomach contents with
overdose is extremely unlikely (e.g. controlled setting such the odor of bitter or burned almonds (Karch 2008). Although
as a hospital or nursing home). This is more feasible to occur not everyone is able to discern this odor, its presence is
when enteric-coated tablets are involved, which do not dis- almost certainly indicative of cyanide intoxication, and may
solve in the stomach, but may stick together to form a small be potentially hazardous in close quarters. In addition, great
mass of tablets (Karch 2008). It is also more likely to happen care is needed if cyanide salts or phosphides, for example alu-
in elderly victims or in other patients where gastric motility minium phosphide, are thought to have been ingested, par-
is slow. The local poisons information service or pharmacy ticularly on an empty stomach, because highly toxic hydrogen
will normally have access to publications or other aids to cyanide or phosphine gas may be released due to reaction
the identification of legitimate and sometimes illicit tablets/ with stomach acid. Additionally, the presence of these and
other volatile materials can lead to cross-contamination of amounts of flexing. Prior to application of the sweat patch, the
other biological specimens unless due precautions are taken. skin should be cleaned using two isopropanol wipes, allowing
Other characteristic odors include the fruity-like odor of alcohol to evaporate completely, otherwise skin irritation can
ethanol and its congeners, the odor of airplane glue (xylene, develop due to isopropanol trapped beneath the sweat patch
toluene), cleaning fluid (halogenated hydrocarbons), carrots (Jenkins 2008). The components absorbed could be eluted
(ethchlorvynol), and garlic (organophosphate insecticides). with water, followed by extraction of XBs before instrumental
In many cases in which death followed the ingestion of nitrite, analysis.
the agent could be identified in gastric content but not in
blood (Blunt 1976). Amniotic fluid
In poisonings involving heavy metals such as arsenic, The increasing use of XBs by expectant mothers has led to
mercury, or lead, specimens of stomach or the small intes- an increased need for pre-natal toxicological testing. For
tine content should also be taken for toxicological analysis. instance, exposure to drugs-of-abuse may result in higher
In addition, illicit XBs are frequently smuggled by ingestion rates of congenital anomalies and neonatal complications.
of balloons or condoms filled with it (the body-packer syn- Therefore, identification of gestational XB exposure may ben-
drome). If these devices burst and an acute XB-mediated efit the newborn in terms of increased vigilance and moni-
death occurs, evidence of these items may be seen in the toring of the infant by medical and social services (Jenkins
gastric content at autopsy (Hassanian-Moghaddam and 2008). Amniotic fluid consists of a filtrate of maternal blood
Abolmasoumi 2007; Kelly etal. 2007; Veyrie etal. 2008; Dinis- that surrounds and protects the embryo during pregnancy. It
Oliveira etal. 2009b). is composed of 99% water and contains cells and fat that may
If intestinal content is sampled, the anatomical source give the liquid a slightly cloudy appearance. Due to its high
from intestinal loops should be notified. water content, little analytical interference exists, making this
specimen easily applied to routinely used toxicological tests.
Sweat At the end of 9 months of gestation, ~ 1.5L of amniotic fluid
Sweat is a fluid excreted from the sweat glands (eccrine and are normally present. Amniotic fluid acts as a fetal excretion
apocrine types). The eccrine glands are widely distributed reservoir, accumulating XBs throughout gestation. Since
at the surface of the whole body while the apocrine glands amniotic fluid is constantly in circulation (being swallowed by
are located in the axillary, mammary, genital, and perianal the fetus, processed, absorbed, and excreted by the fetal kid-
regions. The maximal excretion volume was reported to be neys as urine at rates as high as 50mL per hour) and since the
~ 2L/day in healthy subjects and ~4L/day in trained sport fetus is encapsulated, prolonged exposure to XBs may ensue.
athletes (Suzuki and Watanabe 2005), but the volumes and XBs can enter amniotic fluid by passive diffusion across the
constituents are greatly different according to individuals, placenta and from excretion of fetal urine in the latter stages
types of gland, and various stresses (emotional, physical, of gestation (Gray and Huestis 2007). Another possible route
and thermal) (Sunshine and Sutliff 1996). The sweat analysis of exposure via amniotic fluid is transdermal diffusion, early
started in ~ 1970, and showed that various XBs can be detected in pregnancy when the skin is poorly developed and late in
in sweat (Sunshine and Sutliff 1996). Johnson and Maibach pregnancy when the production of vernix caseosa takes place.
(1971) reported that there is a close relationship between The major limitation of amniotic fluid is sample collection. In
pKa of a XB and its amount of excretion into sweat, and also fact, this sample can only be non-invasively collected at birth
between XB concentrations in sweat and in plasma. However, or as excess specimen from amniocentesis (typically 530mL
collecting sweat is not easy and it is difficult (or even impos- of amniotic fluid is removed) that usually takes place between
sible) to obtain it quantitatively. Therefore, sweat should be the 16th and 20th week of pregnancy (Jenkins 2008). Positive
applied for qualitative toxicological analysis. In the majority toxicological analysis for a specific XB or their metabolites
of cases, the sweat is collected by wiping the skin surface suggests that the fetus has been exposed to these substances
with cotton, gauze, or towel and by using patches attached through maternal blood circulation.
to the skin (Sunshine and Sutliff 1996). Sweat collection is Commonly, this sample is not collected for monitoring
non-invasive and commercially available sweat patches may in utero XB exposure alone (Gray and Huestis 2007; Lozano
be worn for an extended period of time (1014 days or so). etal. 2007). A maternal serum sample obtained at the same
Controlled dose studies concluded that a sweat patch must time may provide complementary toxicological data and help
be worn for a minimum of 24h to collect sufficient amount to assess the relative risk to the fetus. In addition, hair, nail
of XB for analysis (Cone etal. 1994), which itself represents clippings, or meconium could be useful in the interpretation
a limitation. The longer periods of XB excretion into sweat of forensic results.
allied to the possibility of an extended use of collection patch,
enables XBs detection even 14 weeks after single admin- Exhaled air
istration (Inoue et al. 1995). The sweat patch can be worn Analysis of exhaled (expired) air is developing fast since it
on the upper arm (the most common area), lower rib cage enables a non-invasive diagnostic of exposure. Measurement
area, and the upper back (Jenkins 2008). Due to the aggres- of concentrations of volatile XBs in exhaled air by infrared or
sive nature of the adhesive, as a general rule, care should be other devices is essential in roadside testing for ethanol as
taken to ensure that the application area is not subject to vast evidence for prosecution of drunk drivers (Tardif etal. 2004;
Turner etal. 2006; Kamat etal. 2007; Tardif, 2007). Breath- of 0.05mL/min while sleeping, 0.5mL/min while spitting,
testing eliminates the need for taking blood samples, can be and 13mL/min or more while chewing (Crouch 2005). Use
used at the roadside, and the result is immediately available, of acid lemon drops or a few drops of 0.5mol L1 citric acid are
making it possible for police officers to charge the suspect on amongst chemical stratagems adopted to stimulate salivary
the scene without delay. The ethanol metabolite, acetalde- flow, which can result in changes in salivary pH that can alter
hyde, has also been measured in this specimen. Exhaled air the secretion rate of ionizable XBs. Using paraffin wax and
analysis is also valuable in assessing exposure to other XBs Parafilm to stimulate oral fluid production may absorb highly
such as carbon monoxide (Paredi etal. 1999; 2000), hydrogen lipophilic XBs. The oral fluid should be allowed to accumulate
cyanide (Stamyr etal. 2008), and anesthetics (Perl etal. 2009). in the mouth until the desire to swallow occurs before being
Obviously the use of this sample is not possible post-mortem, expelled into the collection vessel. Special attention must be
due to the need to take breath directly from living subjects given if secretion of oral fluid was subjected to stimulation
(Harrison etal. 2003). or not. When the fluid enters the mouth, carbon dioxide is
lost and the pH increases. Dawes and Jenkins (1964) dem-

Saliva/oral fluid onstrated that oral fluid pH is inversely proportional to flow
Since the report in the middle 1950s that XBs were movable rate and the reabsorption of sodium in the salivary tubules.
from blood to saliva, many researchers examined the useful- At faster flow rates, less sodium is reabsorbed in the tubules
ness of saliva analysis, and clarified that XB concentrations on the way from the saliva glands to the saliva outlets in the
in saliva reflected those in blood. Saliva is the excretion mouth and the pH rises. For this reason, unstimulated oral
product originating from three pairs of major salivary glands fluid has a low pH (at low flow rates between 6.07.0, and
(parotid, submandibular, and sublingual), a great number of fairly constant) and stimulated oral fluid has a higher pH (it
minor salivary glands, the oral mucosa, and gingival crevices. can reach as high as 8.0). When the fluid reaches the mouth
Small amounts of cellular debris may also be present. As this it is hypotonic to plasma. Like the liver, kidney, and brain the
excretion product is actually a fluid mixture, the term oral salivary glands, are well supplied with arterial blood. Salivary
fluid seems more appropriate, instead of saliva or whole glands are activated by the autonomic nerves. Generally,
saliva (Malamud and Tabak 1993). The New York Academy of sympathetic stimulation via noradrenaline causes low levels
Sciences meeting on saliva testing, in 1993, agreed to use the of fluid and high concentrations of protein, while parasym-
word saliva for glandular secretions collected directly from pathetic stimulation via acetylcholine induces large amounts
the saliva glands (most often the parotid glands), and oral of fluid secretion.
fluid for fluid collected by placing absorbants in the oral cav- Human saliva normally has a lower pH than human
ity or by expectoration (Malamud and Tabak 1993). Although plasma. Therefore, the oral-fluid:plasma ratios for acid XBs
saliva may be collected from, for example, the parotid gland are generally less than unity while ratios for basic XBs are
by cannulation of the glandular duct, the collection of mixed greater than unity, increasing levels of basic XBs in oral fluid.
whole saliva is normally the only practical alternative. For XBs that have a pKa between 5.58.5, the oral fluid:plasma
A variety of methods are available for oral fluid collection, ratio can vary between stimulated and unstimulated oral fluid.
including spitting, draining, suction, and collection on vari- This is true for many drugs of abuse. For this reason, it is more
ous types of absorbent swabs (Drummer 2006; Gallardo and conservative to use a cut-off value for drugs of abuse in oral
Queiroz 2008). Several devices are also commercially avail- fluid rather than to determine the absolute concentration.
able for on-site analysis, including instruments that provide The most common example given is that of cocaine, which
an electronic readout and hand-held cartridges that require has a pKa of 8.6 (Schramm etal. 1992). The oral fluid:plasma
visual identification. The main advantage of these devices ratios for cocaine can be as higher as 6, when the oral fluid
is that they provide a preliminary XB result within minutes pH varies from 6.5 -8.5.
without the need for sophisticated laboratory screening Different mechanisms of XBs transport are thought to
equipment. The results provided by these devices must be occur, such as passive diffusion through the membrane,
confirmed in the laboratory. Subjects should not brush their active processes against a concentration gradient, filtration
teeth or practice other methods of oral hygiene for several through pores in the membrane and pinocytosis (Gallardo
hours before saliva is collected. and Queiroz 2008). Most of the XBs enter oral fluid by a
Water corresponds to 99% of the oral fluid content. Other mechanism of passive diffusion, which is dependent of the
components such as proteins (0.3% mucins and 0.3% diges- molecular weight (a molecular weight of less than 500Da
tion enzymes, largely amylase) and mineral salts are also favors diffusion), liposolubility, pH and pKa, protein bind-
present. Its pH is 6.8 in resting situations, but an increase in ing, and ionization state (Paxton 1979; Aps and Martens
the salivary flow turns it more basic (approaching the plas- 2005) of the XB, as shown by the HendersonHasselbach
mas pH) as a result of higher osmolarity (Kintz and Samyn (Spihler 2004) equation. Diffusion requires that the XBs (or
2000). All these characteristics are influenced by a variety of metabolites) be lipid-soluble, non-ionized, and unbound.
factors, such as the circadian rhythm, the type of the saliva- Therefore, the concentrations of XBs in oral fluid represent
tion stimulus, hormonal changes, stress, and therapeutic the free non-ionized fraction in the blood. Since these are the
drugs (Aps and Martens, 2005). The total volume of oral fluid forms of the XBs that cross the bloodbrain barrier and affect
produced by an adult may be 1000mL/day, with typical flows performance and behavior, oral fluid is a good specimen for
detecting patient compliance with medication, XB involve- detection of XBs in the hair, sweat, or urine. In general, XBs
ment in driving behavior, fitness for duty, or impairment are detectable in the plasma and oral fluid from the time
of performance for many XBs. However, efforts to use oral that the XB enters the general circulation until approximately
fluid concentrations to predict blood free-XB concentrations four half-lives after exposure. Another limitation drives from
or degree of performance impairment in individuals have the fact that the majority of the XBs exposure are from the
not reached the accuracy of blood measurements. Without oral route. Ingested XBs as well as those that can be smoked
knowing the instantaneous oral-fluid pH, oral-fluid XB con- or snorted may be detected in high concentrations in oral
centrations may not be extrapolated to give blood XB con- fluid following recent use, due to residual amounts of XBs
centrations. The protein binding of XBs in plasma is mainly remaining in the oral cavity. Therefore, the analytical results
to albumin. Oral-fluid mucoproteins have very little binding are not accurate since XBs concentrations found in the oral
capacity for XBs, although oral fluids may contain albumin fluid do not reflect their blood concentration. In cases that
from the gingival crevicular fluid. involve these routes of administration, 24h should elapse.
The advantages of oral-fluid XB testing are various. In Jenkins etal. (1995) showed that the oral fluid:plasma ratio
principle, oral-fluid XB concentrations can be related to for smoked heroin was 100400-times higher than when her-
plasma free-XB concentrations and therefore to the phar- oin was administered intravenously. After smoking heroin,
macological or toxicological effects of XB. Second, oral- heroin was detected in oral fluid for up to 24h, compared to
fluid collection is non-invasive, easier than venepuncture, up to 30min after heroin was intravenously administered.
can be done on-site under direct supervision without loss The same authors reported that after smoking cocaine the
of privacy and can be done by the donors themselves in oral fluid:plasma ratio was 300500-times higher than that
most situations (Spihler 2004). In consequence, the risk of found after cocaine was administered intravenously. The
an invalid specimen being provided or sample adultera- pyrolysis product of cocaine, anhydroecgonine methyl ester,
tion and/or substitution (which are likely to occur in urine was detected in oral fluid collected after smoking cocaine,
analysis) is reduced. In addition, oral fluid monitoring may but not in plasma. Similarly, cannabinoids found in oral fluid
be especially important when multiple serial samples are almost totally result from oral deposition of cannabinoids
needed or when XB concentrations in children are required from smoked marijuana rather from secretions or diffusion
(Kim et al. 2002). The feasibility of detecting XBs in oral into oral fluid (Ohlsson et al. 1986). ONeal et al. (2000)
fluid samples obtained from impaired drivers was first reported codeine oral fluid:plasma ratios of 752580 in the
investigated by Peel et al. (1984). Authors found that the first 1530min after dosing and of 13344 for several hours
presence of XBs in oral fluid correlated well with officer after oral administration of liquid codeine phosphate, despite
judgments of driving while intoxicated. This was confirmed decontamination efforts by having the subjects brush their
in a comparison of saliva testing to urinalysis in an arrestee teeth and vigorously rinse their mouth prior to oral fluid col-
population (Yacoubian et al. 2001) and in drugged driv- lection. The formation of oral mucosa depots of XBs, which
ers (Steinmeyer et al. 2001). Several methods have been are rapidly absorbed into the blood circulation, is used for
described to detect opiates, cannabinoids, amphetamines, XB administration. Sublingual or buccal absorption of XBs,
cocaine, benzodiazepines, ketamine, hydroxybutyric such as nitroglycerine, buprenorphine, or fentanyl, has the
acid, antibiotics, analgesics, cyanides and other tobacco advantage of very rapid delivery that bypasses the liver and
compounds, sildenafil and many other XBs (Gallardo and gastrointestinal first-pass metabolism. XBs administered by
Queiroz 2008). this route also produce large concentrations of the parent XB
However, like any fluid from human subjects, oral fluid in oral fluid, but with a short detection window, since XBs
may transmit infectious agents and should be handled are rapidly absorbed (Spihler 2004).
with the appropriate universal precautions for human bio-
logical fluids. Oral fluid contains mucopolysaccharides and Feces
mucoproteins that make it less fluid (more viscous) and less After the kidney, the second most important route of elimi-
easily poured or pipetted than urine or plasma. Routine nation of many XBs is through feces. Nevertheless, fecal
dilution with aqueous collection buffer is advocated by excretion of XBs is a complex process that is not as well
some authors to minimize this problem. Some XBs, medi- understood as urinary excretion (Klaassen 2008). The main
cal conditions (when the donor is unconscious or sedated), mechanisms contributing to the presence of XBs in feces are
or anxiety can inhibit oral-fluid production and cause dry the following:
mouth. Therefore, oral-fluid may not be available from all
individuals at all times or they can produce insufficient a. Incomplete absorption, as occur in paraquat intoxications
amounts of sample for analysis. In addition, reliable oral- due to its hydrophilic nature (Dinis-Oliveira etal. 2008);
fluid collection requires a co-operative individual and even
b. Intestinal secretion, which likely occurs by passive diffu-
then is not without problems. Finally, because oral fluid XB
sion out of enterocytes or via exfoliation of intestinal cells
concentrations depend on plasma XB concentrations, XBs
during the normal turnover of this epithelium; and
that have a short plasma half-life and are cleared rapidly
from the body are detectable in oral fluid for a short time c. Biliary excretionperhaps the most significant source
only (Spihler 2004). This is a potential limitation over the contributing to the fecal excretion of XBs, and is even
more important for the excretion of metabolites. meconium are generally higher than in urine because of its
P-glycoprotein is considered the main transporter accumulation over several months of gestation (Ostrea 2001;
involved, as previously proved for paraquat (Dinis- Ostrea etal. 2001). One major advantage of meconium is a
Oliveira etal. 2006b). relatively wide window for sample collection. Unlike urine,
which allows the detection of fetal XB exposure for only 23
The analysis of feces is rarely performed in clinical chem- days before birth, meconium extends this window to ~ 20
istry, analysis being restricted mainly for pharmacokinetic weeks pre-partum (Moore etal. 1998; Kintz and Samyn 2000),
and metabolism studies (Sorg etal. 2009) or if the question and therefore history of in utero XB exposure during the
of ante-mortem XB leakage from ingested packets is raised. second and third trimesters is highly possible. These factors
Unlike plasma, urine, and other fluid samples, feces are not make meconium an optimal matrix for identifying in utero
homogeneous, and thus it is often necessary to analyze the exposure as it is considered a preserved record of the ultimate
whole sample or homogenize the whole sample and prove exposure by the fetus. After birth, meconium is excreted by
that the fraction taken for analysis is representative of the the neonate several times a day for the first 15 days post-
whole (Flanagan etal. 2007). partum. Viable analysis appears to be optimal via collection
within 72h, since in the later stages of meconium excretion
a matrix of meconium and feces is produced. In extremely
Meconium
low birth weight infants, which is of particular interest in the
Prenatal exposure to XBs is an ongoing concern, with signifi-
XB exposed neonatal population, median age of first stool
cant impact on neonatal health and development (Meeker
is 3 days, with 90% of infants passing their first stool by day
and Reynolds 1990; Dusick et al. 1993; Gingras et al. 2004;
12 (Verma and Dhanireddy 1993). For these reasons, the
Wouldes et al. 2004; Hurd et al. 2005; Smith et al. 2006).
possibility of sample collection beyond 48h post-natally is
Meconium, the first fecal matter passed by a neonate, has
a remarkable advantage of this matrix (Ostrea 2001; Ostrea
recently been given much attention because it is a useful
etal. 2008). Issues relating to the screening and confirma-
specimen to determine fetal exposure to XBs, it is easily
tion of most drugs of abuse in meconium have been reviewed
collected and non-invasive, large amount of sample can be
(Lewis etal. 1994; Moore etal. 1995a; b; 1998; Le etal. 2005;
collected and can provide information regarding long-term
Lopez etal. 2007; Gallardo and Queiroz 2008).

exposure. Meconium, in comparison to urine, is easily col-
Most post-mortem toxicology laboratories are not cur-
lected. For newborn urine collection, bags are taped to the
rently performing meconium analysis. While potentially
babies, and these often become detached or urine is spilled.
useful, there are several limitations that must be considered.
Meconium is simply scraped from a diaper and placed in the
Because meconium forms layers in the intestine as it is being
collection vial (Jenkins 2008). Post-mortem meconium analy-
deposited, it is not a homogeneous specimen (Karch 2008).
sis can also constitute additional help in the medico-legal
As with other non-homogeneous specimens, such as gastric
practice, in cases of sudden infant death syndrome and of
content, it is important that all available specimens are col-
unexplained late fetal death. It is identified most commonly
lected and thoroughly mixed before aliquoting. Meconium
by its dark green/black color because of the presence of bile
analysis is more labor intensive and requires more time to
and a lack of the odor of regular feces.
analyze than urine. Often the sample size obtained is small,
It is a highly complex matrix consisting of water, muco-
and the concentration of XBs found in meconium is rela-
polysaccharides, bile salts, bile acids, lanugo (fine neonatal
tively low (Jenkins 2008). However, advances in technology
hair), desquamated epithelial cells from the gastrointestinal
have improved the sensitivity of testing procedures, so this is
tract and skin, lipids, proteins, as well as the residue of swal-
increasingly of less importance.
lowed amniotic fluid (Chan etal. 2004a; b; Gareri etal. 2006).
Fetal swallowing is thought to be the mechanism by which
XBs are concentrated in the meconium, since as the fetus Liver
releases urine into the amniotic fluid, any excreted XBs and The liver has been ranked as the primary solid tissue for
metabolites are then swallowed and ultimately deposited use in post-mortem toxicology, and often the XB analysis,
into the meconium (Browne etal. 1992; Ostrea etal. 2006). resulting from this tissue, complements the blood toxicol-
In addition to this mechanism, fetal exposure is a product ogy data (Luckenbill et al. 2008; Gronewold et al. 2009;
of maternal consumption, metabolism and elimination, pla- Vance and McIntyre 2009). For many XBs, especially those
cental transfer and metabolism, and also fetal metabolism with chemically basic character (e.g. alkaloids), higher
(Gallardo and Queiroz 2008). concentrations can be found in the liver comparatively to
The timing of meconium formation has been variably blood (Jenkins 2008). Most case reports illustrating toxicol-
reported in the literature. The assertion that meconium ogy findings include concentrations of the XBs in liver. It is
begins to form at ~ 12 weeks of gestation is likely to be the also the favored specimen when blood is not available due
most accurate, since it is at this time that fetal swallowing of to exsanguination, fire, or decomposition. As a specimen,
amniotic fluid begins (Kwong and Ryan 1997), and the for- liver also has the advantages that it is the major metabolic
mation of meconium has been evidenced at this time period organ, sufficient quantities are available for analysis, and it is
by the presence of cocaine found in the meconium of early reasonably homogeneous and relatively unaffected by post-
gestational fetuses (Ostrea etal. 1994). XBs concentrations in mortem redistribution or post-mortem diffusion compared
with blood. Nevertheless, XBs concentrations in the lobe Lung

proximal to the stomach may artificially increase by post- High concentrations of XBs are frequently found in lung tis-
mortem diffusion in cases of oral overdose. Therefore, use sue, especially after poisoning by intravenous or inhalatory
of tissue from deep within the right liver lobe is preferred routes. Depending on the properties of a XB, concentrations
(Figure 11) (Pounder and Smith 1995; Pounder etal. 1996b). in lung tissue can be higher than in liver (Dinis-Oliveira
Bile should be collected prior to the liver specimen, to pre- et al. 2008; 2009a). When solvent abuse or an anesthetic-
vent contamination. Liver has been found to be particularly mediated death is suspected, lung and brain specimens
suitable to determine tricyclic antidepressants and many must be available for toxicological investigation. In addi-
other XBs that are highly protein bound (Apple and Bandt tion, air may be collected directly from the trachea with a
1988; Apple 1989; Moriya and Hashimoto 1999c; Davis etal. syringe and injected into a sealed vial to be used for head-
2001). It is useful for the phenothiazine neuroleptics, which space analysis. In paraquat intoxication-related deaths,
have a large dosage range (Robinson etal. 1974; Apple and lung should always be analysed (Dinis-Oliveira etal. 2006a;
Bandt 1988; Anderson etal. 1999; Davis etal. 2001; Yarema 2008). Paraquat mainly accumulates in the lung (pulmonary
and Becker 2005; Kirkton and McIntyre 2006; Wenzel etal. concentrations can be 610-times higher than those in the
2006). Analysis of a liver tissue specimen may help to dif- plasma), where it is retained even when blood levels start
ferentiate acute overdose from therapeutic use of XBs with to decrease. Accumulation occurs against a concentration
a narrow dosing window. In cases where the concentration gradient, through the highly developed polyamine uptake
of basic XBs in blood is high and ratios of liver-to-blood XBs system. A negative result in blood is particularly common,
concentration exceed 10, a XB fatality is strongly suggested lung being soaked in paraquat (Dinis-Oliveira etal. 2006a;
if no other interceding cause of death is present (Karch 2008). Pounder etal. (1996b) observed that amitriptyline and
2008). Smaller ratios, even with high heart blood concen- paracetamol added to the stomach of XB-free cadavers diffuse
trations, tend to suggest a greater potential for post-mortem into the left lung, left lobe of the liver, the spleen, and into the
redistribution of XBs into the blood. Due to the lipid-soluble pericardial fluid. To reduce the problem of post-mortem XB
nature of many XBs and the high lipid content of the liver, diffusion from the stomach and gastrointestinal tract, it has
long-term sequestration for many XBs in this organ is likely been recommended that lung should be sampled from the
to occur, originating with a longer half-life than the tradi- apex rather than the base (Figure 12) (Pounder etal. 1996a).
tional half-life in circulating blood, and this should be taken
into account when interpreting very low concentrations of Kidney
XBs in the liver (Jenkins 2008). Limitations of utilizing liver A kidney specimen can be a useful sample for XB identifi-
include the necessity to produce a homogenate prior to cation since most XBs and metabolites are excreted into
extraction, matrix effects observed with routine analytical urine and therefore will pass through the kidneys. It is par-
techniques, and lack of a database to aid the interpretation ticularly important in cases of heavy metal poisonings due
of XB concentrations. to its capacity of accumulation of these XBs (Yilmaz 2002;
A B
C D
Figure 11. Liver should be collected from deep within the right liver lobe (ad; dashed line).
Triunfante etal. 2009). In addition, structural damage to the tified in numerous cases of decomposed, dried skeletal
kidney due to heavy metal or ethylene glycol exposure may muscle (Manhoff etal. 1991);
be documented histologically, supporting the toxicological d. Sampling is possible from various sites away from XB
analysis (Post etal. 1984; Scott etal. 1987; Sugita and Tsuchiya
reservoirs in liver, lung, or gastric content. Normally,
1995; Barregard etal. 1999; Debacker etal. 2000; Rumbeiha specimens of iliopsoas muscle (right or left of the lumbar
etal. 2000; Alonso etal. 2005; Ferrari and Giannuzzi 2005; portion of the spine) are collected (Figure 14) (Schloegl
Takahashi etal. 2008). etal. 2006a; b); and
Spleen e. Concerning the homogeneity of the tissue, different
Spleen, an organ rich in blood, is useful for the analysis of theories exist (Christensen et al. 1985; Garriott, 1991;
XBs that bind to hemoglobin, such as carbon monoxide and Williams and Pounder 1997).
cyanide. Frequently, in fire deaths where extensive charring
is present, spleen may be the only useful specimen available Interesting data on XB concentrations from muscle speci-
to perform these assays. mens were provided by Langford and coworkers (Langford
and Pounder 1997; Langford etal. 1998) and Williams and
Cardiac muscle Pounder (1997). Twelve different muscle samples collected
A cardiac muscle specimen can also be a useful sample for from overdose cases (n=11) as well as from cases with
XB identification. Although no specific roles exist to obtain chronic therapeutic use (n=3) were analyzed in the first two
heart muscle, collection of samples from the left ventricle reports. These results were compared with each other and
has been reported by several authors (Figure 13) (Hikiji etal. with the XB concentration in corresponding femoral blood.
2008; Luckenbill etal. 2008; Thiblin etal. 2009). The muscle-to-blood ratio was influenced by the time lapse
between XB exposure and death as well as by the volume of
Skeletal muscle distribution of the XB. The within-case variability observed
Skeletal muscle has several advantages and many potential in muscle specimens supports the opinion that XB analysis
applications in post-mortem Forensic Toxicology. It meets on skeletal muscle is rather qualitative than quantitative in
many of the criteria of an ideal forensic specimen: nature. The same conclusion was provided by Williams and
Pounder (1997), analyzing a series of eight fatal XB over-
a. It is present in large quantities (often represents the doses. Studies performed by Garriott (1991) showed that XB
greatest single mass of XB in a body and will therefore concentrations in thigh muscle reflect XB concentrations in
represent a greater body burden of XB than any other blood for many common basic XBs and ethanol, except in
tissue mass, ~ 30 Kg, or 43%, of a 70-Kg person); cases of an acute XB death where muscle XB concentrations
may be lower than blood due to inadequate time for tissue
b. Almost always available even in putrefaction, trauma,
equilibration. The analysis of thigh muscle was proposed to
and burning;
be especially useful in cases where XBs suspected of under-
c. Less affected by decomposition than blood and internal going post-mortem redistribution or diffusion are detected in
organs and therefore it may be useful as an indicator of the heart blood (Garriott 1991). Christensen etal. (1985) also
post-mortem blood concentrations (Garriott 1983; 1991; suggested to collect the muscle extremity, where possible,
Christensen et al. 1985). Surprisingly, even cocaine, as XBs concentrations in other muscles, such as abdominal
which is known to be unstable in blood, has been iden- muscle, increase with time while remaining constant in thigh
muscle.
The potentially useful data that may be obtained from
the analysis of skeletal muscle have prompted some toxi-
cologists to recommend skeletal muscle to be collected in
all cases where XBs may be implicated in the cause of death
(Christensen etal. 1985). One limitation of skeletal muscle
is that its homogenization, prior to analysis, is very diffi-
cult, though it is required to ensure complete XB extraction
(Williams and Pounder 1997). As more laboratories analyze
skeletal muscle leading to the availability of additional data
to aid in the interpretation of results, its potential advantages
will outweigh the limitations.
Adipose tissue
As for skeletal muscle, adipose tissue is not frequently ana-
lyzed due to the difficulty in reliably extracting XBs and
because of substantial variability in parent compound/
Figure 12. Lung should be sampled from the apex rather than the base of metabolite concentrations from one site to another. However,
the right lung (dashed line). these tissues represent the greater mass of a body and a greater
A A
B
Figure 14. For skeletal muscle, normally, specimens of iliopsoas mus-

cle (right or left of the lumbar portion of the spine) are collected (dashed
line).
Figure 13. Collection of cardiac muscle from the left ventricle has been
suggested (dashed line).
mortis are essentially the same as those without it (Levisky

body burden of XB than any other tissue. Until now, there is etal. 2001). Also, since adipose tissue is poorly vascularized,
no guidance from which part of the body should this sample and only ~ 2% of the blood supply is distributed throughout
be obtained, but abdominal subcutis adipose tissue has been this layer, any contribution of XBs coming from blood vessels
considered in the majority of forensic works (Schloegl etal. would be minimal. In an animal study, higher amounts of
2006b). Adipose tissue acts as a reservoir for many lipophilic fatty ethyl esters, as post-mortem markers for ethanol intake,
XBs (Sorg etal. 2009). For instance, 9-tetrahydrocannabinol were present in adipose tissue, compared to liver (Salem etal.
has an avid affinity for it with concentrations ~ 200-fold higher 2001; Refaai etal. 2002). As for skeletal muscle, adipose tissue
than that in circulating blood (Chu 2002). Although few may be useful for post-mortem examination of injection sites,
reports on XB detection in post-mortem adipose tissue have due to higher XB concentration.
been published (Levisky etal. 2000; 2001), some important
conclusions were ascertained. It was reported that XBs identi- Nasal, oral, and skin swabs
fied in the adipose tissues of cadavers are there because of Usually, cotton tips are used to collect nasal, oral, and skin
ante-mortem deposition and not post-mortem redistribution swabs by carefully rubbing over the suspected areas (Skopp
(Levisky etal. 2000; 2001). To reach these conclusions, the 2004). The tip may be moistened with methanol or ethanol
authors examined tissues with and without livor mortis. Livor prior to sampling. The swabs should be placed into a transport
mortis (also referred to as hypostasis) and post-mortem livid- tube and sealed. Skin swabs for XB analysis should be taken
ity are terms used to describe the staining of the skin surface from sites that had been covered by clothes to minimize exter-
and internal organs by the settling of blood, under the influ- nal contamination (Skopp 2004). Nasal and oral swabs allow
ence of gravity, after death (Henssge etal. 2002). Livor mortis the simple detection of a XB. However, a positive finding does
can be considered a form of vascular post-mortem redistri- not prove that the XB had been intranasally administered or
bution involving the movement of blood within the vascular orally ingested because pulmonary edema fluid may be the
compartments. In this case, even though there is obvious source of the material tested. For example, the simple detec-
redistribution of vascular components, including red cells tion of cocaine in a nasal swab does not prove that the XB
and hemoglobin, there was no evidence of the infiltration or was snorted. Any fluid secreted by the body, including sweat,
diffusion of XBs from vascular sources outside the adipose vaginal fluid, and nasal secretions, will contain some concen-
layer: the cocaine concentrations in the tissues with livor tration of the XB. The same is true for skin swabs. Although
skin swabs have became very popular in roadside testing in may indicate attenuation of use, this finding being useful in
some countries, in the last decase (Skopp and Potsch 1999), cases in which a relapse in XB use could have been related
this specimen is of limited value in post-mortem toxicology. to death.
Traditionally, hair, along with fingernails, has been the
Keratinized tissues specimen of choice in determining chronic heavy metal
In recent years, keratinized tissue such as nails and hair have poisoning such as arsenic, mercury, and lead (Kintz et al.
received considerable attention, since these samples may 2006; 2007b). Keratin, found in large amounts in hair and
provide retrospective information that far exceeds that from nails, is an excellent source of sulfhydryl groups to which
blood or urine. heavy metals bind to form covalent complexes. Since 1979,
when morphine was first detected in a hair specimen from a
heroin user (Baumgartner etal. 1979), numerous other XBs
Hair
have been identified in hair besides heavy metals (Uematsu
Hair possess a shaft and root and is constituted by proteins
1993a; b; Kintz 2004; 2007; Kintz etal. 2007a). In drug-facil-

(6595%, mainly keratin), water (1535%), lipids (19%),
itated crimes, the detection of a particular compound, such
and minerals (0.250.95%) (Wennig 2000). A rich capillary
as hydroxybutyric acid (Kalasinsky etal. 2001; Goulle etal.
system, which provides the growing hair with the necessary
2003; Kintz etal. 2003; 2004; 2005; 2008), in hair, has been
metabolic material, surrounds the hair follicle (Pragst and
used to document the exposure, but usually a negative find-
Balikova 2006). The growth rate is dependent, to some extent
ing cannot exclude an exposure (Cheze etal. 2005). If a single
on individual factors, such as age, hair care, and environmen-
exposure to a XB is suspected, as in drug-facilitated sexual
tal factors, anatomic region, sex, health conditions, pigmen-
assault, but the suspected XB is not detected in blood or
tation, and culture or race (Wennig 2000). Growth rates are
urine, waiting for 12 months for head hair to grow and then
~ 0.61.4cm and 0.6cm per month for scalp and pubic hair,
performing segmental analysis may reveal the presence of the
respectively (Kintz 2004). Beard hair is also a suitable speci-
XB (Kintz etal. 2003). Other important applications are the
men for analysis. This type of hair grows at ~ 0.27mm per day,
detection of in fetal utero exposure, resulting from maternal
and therefore can be collected on a daily basis with an elec-
XB use, and the use of XBs in cases of child abuse (Klein etal.
tric shaver. Therefore, significant differences in the anagen
1992; 1993; Koren, 1995; Koren and Klein 1997; Koren etal.
(active growing)/catagen (transition)/telogen (resting) stages
1998; 2002; 2008; Klein and Koren, 1999; Boroda and Gray
of the hair growth cycle, and in the respective growth rate,
2005). The use of hair in workplace XB testing is controversial
exists (Mangin and Kintz 1993), justifying that the length, the
due to issues such as environmental contamination (Wang
color, an obvious cosmetic treatment, and the collection site
and Cone 1995; Cone 1996; 1997; 2001), washing techniques
of the specimen should be documented in order to facilitate
(Blank and Kidwell 1993; 1995), sex or ethnic bias, the dif-
the interpretation of the results. For instance different XB
ficulty in performing quantitative analysis (Welch etal. 1993),
concentrations between pubic or axillary hair and scalp hair
and establishing cut-off concentrations (Kintz 1995; 2004;
were found (Han etal. 2005) and some XBs are susceptible to
Kintz and Mangin 1995; Kintz etal. 1995).
degradation by cosmetic treatment and/or by sunlight (Skopp
Although the precise mechanisms involved in the incor-
etal. 1997; 2007).
poration of XBs into hair have not been completely clarified,
Hair has a long history as a useful specimen in Forensic
Toxicology, especially used as a supplementary tool to dis- three processes have been proposed (Blume-Peytavi et al.
2008; Jenkins 2008; Karch, 2008):
close or confirm previous XB use (Karch 2008). Hair is a
unique material for the retrospective investigation of chronic
a. Passive diffusion from blood capillaries into the growing
exposure since it provides a longer window of detection
cells at the base of hair follicle;
compared to blood or urine, and is less invasive to collect.
Long scalp hair may provide retrospective information of the b. Diffusion from sweat or sebum secretions; and
previous 57 years (Daniel etal. 2004). In addition, analysis of c. Passive exposure to XBs from an external source, such as
hair for XBs is not more difficult or challenging than testing from smoke or dirty hands, and secondary to dissolution
in other matrices, since the application of analytical methods of the XB into the XB-free sweat.
and instrumental approaches is, in most cases, quite similar,
regardless of the initial sample preparation. Hair analysis has It is virtually impossible to distinguish between the presence
also been successfully applied on exhumed human bodies. of XBs derived from these two latter mechanisms and that
In some circumstances, post-mortem specimens may not be proceeding from real administration, which is explained by
available, and hair, due to its resistance to decay, could be the the fact that the XBs are in an aqueous moiety, enhancing
only sample available for testing. Hair may also survive longer their incorporation, and because of the fact that hair is very
after burial than other tissues (Kintz 2004). Interpretation of porous and can increase its weight up to 18% by absorbing
positive findings can be augmented by the segmentation of liquids, which facilitates incorporation of XBs into the hair.
the hair strands to assist in determining the time of expo- This is the reason why environmental exposure is sometimes
sure and define historical XB use or changes in XB habits (or called the stumbling block of hair testing. Incorporation of
abstinence) (Smith 1964; 1976; Kintz et al. 2006; 2007b). A XBs is also affected by the melanin content of the hair and by
decrease in XB concentration in the proximal sections of hair the substances lipophilicity and basicity. For instance, basic
XBs incorporation in pigmented dark hair can be ~ 10-fold This procedure attempts to avoid misalignment of a hair sam-
higher than in non-pigmented grey hair (Pragst and Balikova ple necessary for segmental analysis (Flanagan etal. 2007).
2006), a fact that has been suggested to be the consequence The sample should be stored in a tamper-proof envelope at
of XBs binding to melanin (Rollins etal. 2003; Mieczkowski room temperature until analyzed (Jenkins 2008). Putting hair
and Kruger 2007). After incorporation, XBs are not further into folded paper should be avoided, particularly in the pres-
metabolized. ence of plucked hair, since the sticky hair roots will become
There are some recommendations about hair specimen fixed to the porous surface of the paper, and the strands will
collection (Bost 1993; Henderson 1993; Miller et al. 1997; break at variable distances from the root (Karch 2008). The
Harkins and Susten 2003; Cheze et al. 2004; Flanagan and size of the sample to be collected is dependent on the purpose
Connally 2005; Flanagan et al. 2005; Madea et al. 2007). If of the analysis. If a segmental analysis is desired, hair from a
possible, hair should be collected from the posterior vertex 12cm area will typically yield ~ 50mg of hair/cm segments,
region of the scalp or the back of the skull, where the average which is the amount used for many GC-MS or LC-MS methods
hair growth rate is fairly constant (Figure 15), and the hair is reported. Additional samples have to be collected if several
less subject to age- and sex-related influences, and has been analyses with different extraction techniques are desired. If
extensively studied. In addition, on the scalp of an adult, ~ 85% the time for exposure is not an issue, smaller hair samples
of the hair is in the anagen phase and the remaining 15% is in (100200 g) may suffice (Karch 2008). Deep freezing of hair
the quiescent stages (catagen and telogen) at any time phase samples have been shown to result in low XB concentrations,
(Henderson 1993). Cut hair is usually preferable since hair which is suggested to be due to XB diffusion from damaged
roots, often containing high amounts of XBs resulting from keratin matrix during melting. If scalp hair is not available
an acute intake, are then excluded. In cases with a suspicion of (or if it is excessively bleached or permed), alternative hair
a recent poisoning, analysis of plucked hair may be rewarding, specimens may be collected from the pubic or axillary sites
since there is an interval for most XBs during which cut hair (Jenkins 2008). Hair contaminated with XB containing blood,
may all be negative, but where the intradermal portion of the vomit, or putrefaction fluid must not be taken, for it is well
hair may harvest traces of the XB. Post-mortem, it is strongly known that absorption of water or aqueous liquids are very
recommended to take a hair sample prior to autopsy. The hair rapid in enabling XBs to enter the hair. Alternatively, hair
sample should be firmly tight together and tied with cotton, should be washed with water and then dried before sam-
before being cut as close as possible to the scalp, making sure pling. It is wise to make a note that such contamination has
the scissors are leveled with the scalp. Still holding the sample occurred if it turns out that the blood contains high levels of
tightly, the cut root ends of the sample must be aligned and XBs. Even sophisticated washing techniques are not capable
carefully placed flat on a piece of aluminium foil, that is folded of completely removing XBs from hair that is derived from
once or twice, with the cut root ends projecting ~ 15mm these sources (Romano etal. 2001; 2003), and therefore spe-
beyond the end of the foil (Figure 15) (Flanagan etal. 2007). cific metabolites of the XBs must be searched for. This is of
A B
Figure 15. Hair should be collected from the posterior vortex region of the scalp or the back of the skull as close as possible to the scalp. The hair sample
should be firmly tight together and tied with cotton (a). Still holding the sample tightly, align the cut root ends of the sample and carefully place flat on
a piece of aluminium foil, that is folded once or twice, with the cut root ends projecting ~ 15mm beyond the end of the foil (b). Mark the root end of the
foil and fold the foil around the hair and pinch tightly to keep in place. Fold the foil again in half lengthwise. (a) and (b) Adapted from Flanagan etal.
(2007).
particular importance in the case of XBs that are likely to be in even potentially longer than hair, and therefore this matrix
the environment because of the way they are consumed, such has the potential to be a useful source for information about
as cannabinoids and cocaine, where at least one metabolite the XB exposure history of the decedent, especially when the
should be detected (Cone etal. 1991; Uhl and Sachs 2004). scalp hair is too short or not available as a result of alopecia
This may present a problem, because normally the metabo- totalis or shaving of many body parts (Caplan and Goldberger
lites are more polar XBs, and have less affinity for hair matrix 2001; de la Torre etal. 2004; Jenkins and Engelhart 2006; Lech
constituents (Cone etal. 1991; Uhl and Sachs 2004). 2006). Also in common with hair, care needs to be taken to
ensure that external contamination is avoided or at least
Finger and toe whole nails or clippings considered in any interpretation of results using these speci-
In 1984, the presence of methamphetamine and amphetamine mens. Unlike hair, nails do not contain melanin, and this may
was demonstrated in nail clippings from habitual users, sug- reduce XB incorporation. In addition, the variable and slower
gesting the usefulness of nails as forensic material (Suzuki etal. growth rate of nails, especially toe nails, as compared to hair,
1984). Since then, fingernails proved to be valuable specimens makes segmental analysis, and hence interpretation, hardly
in suspected heavy metal poisoning, such as thallium, arsenic, possible (Miller etal. 1997; Lemos etal. 2000; Maurer 2000;
or lead (Chen etal. 1999; Mandal etal. 2004; Brima etal. 2006; Palmeri etal. 2000; Caplan and Goldberger 2001; de la Torre
Lech 2006; Kile etal. 2007; Sanz etal. 2007). A number of other etal. 2004; Jenkins and Engelhart 2006; Lech 2006; Payne-
trace elements showed considerably more concentration in James etal. 2007; Mari etal. 2008).
nails than in urine or blood (Karpas 2001; Karpas etal. 2005a; Ante-mortem, all finger- and toe-nails clippings should be
b; 2006). Other XBs detected in nails include cocaine and collected and combined. Nail clippings from donors using
its major metabolites such as benzoylecgonine, ecgonine Teflon-coated stainless steel scissors will be desirable to
methyl ester, norcocaine and cocaethylene, 6-acetylmorphine, reduce contaminations. In post-mortem work, whole nails
morphine, codeine, hydromorphone, oxycodone, hydroco- should be lifted from the fingers or toes. Some authors con-
done, 9-tetrahydrocannabinol, and 11-nor-9-carboxy-9- sider that toenails are better than fingernails because they
tetrahydrocannabinol (Pichini et al. 1996; Engelhart et al. are less exposed to external contamination. Whole nails or
1998; Palmeri etal. 2000; Engelhart and Jenkins 2002; de la clippings can be stored at room temperature for very long
Torre etal. 2004; Ragoucy-Sengler and Kintz 2005; Gray and periods, including years, without major degradation of incor-
Huestis 2007; Ali etal. 2008). porated XBs, enabling re-analysis. The stability of XBs in nails
Nails grow according to two different directions, length and makes their analysis a good tool for post-mortem investiga-
thickness. Length growth rates of nails were reported to be tions, especially when it is impossible to perform other tests
35mm (Hamilton etal. 1955) and 1.1mm (Bean 1953; 1980) because of the lack of common body fluids or when decom-
per month for the fingers and toes, respectively. The thicken- position of the remains can produce false results (e.g. nega-
ing rate is constant and slow, with a mean value of 0.027mm/ tive tests due to the instability of analytes in body fluids or
mm length (Johnson and Shuster 1993). Nevertheless, many false positives from low specificity screening tests because of
different values are given in the literature by several authors the presence of interferences) (Garside etal. 1998). Once XBs
and many factors can influence it. Growth is slowed with are incorporated into nails, levels remain relatively constant
increasing age, cold climatic conditions, disease, and mal- since fluctuations are not expected to occur due to changing
nutrition, but it is faster in nail-biters. Even if there is consid- of body metabolic activities, unlike the blood (Takagi etal.
erable individual variation, the growth of the nail of the third 1988). Nails that have been polished should not be sampled
digit seems to be greater than that of the others. No differ- because elements added during the nail polishing may
ences seem to occur between the rate of nail growth on the become an external element source (Sukumar 2006).
right and left hands or between men and women (Singh etal. In infants born from drug abusing mothers, nail analysis
2005). It requires ~ 36 months for a whole nail to replace may offer some advantages over hair (Skopp and Potsch
itself (Gupta etal. 2005). It is usually considered to be a third 1997). Compared to hair, that is not always present in the
of average hair growth rate. scalp of newborns or lost in significant amounts during the
Relatively little is known about the mechanisms of uptake first months of life, and the analyst may encounter some
and retention or XBs and metabolites in nail, making the resistance to collect it for cosmetic reasons, nails have the
interpretation difficult. Some literature data has shown that substantial advantage of always being present and con-
XBs are incorporated into nails by a double mechanism sidered discardable material (Mari et al. 2008). Finally,
(Palmeri etal. 2000): the well-known drawback of nails analysis, that is external
contamination by manipulation of XBs, is definitely unlikely
a. Deposition into the root of the growing nail via the blood in the case of a baby (Mari etal. 2008). On the other hand,
flow in the nail matrix; and in order to reflect fetal life, all the nail clippings of the first
b. Incorporation via the nail bed during growth from the 3 months are needed, and these samples are not always
lunula to the beginning of the free margin. gladly and continuously collected by the mother and, hence,
not easily gathered (Mari etal. 2008). Nails are formed dur-
Like hair, finger- and toenails accumulate XBs during long- ing the last trimester of pregnancy and are, thus, supposed
term exposure, providing a retrospective window of detection, to reflect the exposure only in this period. However, similarly
to hair, incorporation of XBs in the nails of the fetus might pleural cavity and placed in a 30-mL-capacity plastic tube
be mediated by the fetal blood and/or by the amniotic fluid. containing sufficient sodium fluoride to give a concentra-
Inasmuch as amniotic fluid is formed since the early stages of tion of ~ 1% (Jones etal. 1999). Sims etal. (1999) showed that
gestation, it is not impossible that the presence of XBs in nails in 21 cases involving 20 different XBs, XB concentrations in
is related to a XB exposure also during the first two trimesters pleural effusions were compared with those determined from
(Mari etal. 2008). blood specimens. Marked differences could be observed in
In a case of sudden infant death syndrome, cocaine was cases involving ethanol, carbon monoxide poisoning, and
detected in nail clippings taken at autopsy from a 3-month- flunitrazepam. In 70% of the cases, reasonable coincidence
old infant, indicating a pre-natal XB exposure (Engelhart with pleural effusion/blood ratios in the range of 0.41.6 was
and Jenkins 2002). Owing to the vast consequences of this found. XB levels in pleural fluid were also compared with
problem and to the importance of opportunely anticipating concentrations in corresponding liver tissue. The authors
the onset of withdrawal symptoms in newborns, a number suggested that bloodstained pleural fluid appears to be a suit-
of bioanalytical procedures for monitoring drug abuse dur- able alternative material for estimation of XB levels in cases of
ing gestation has been proposed, involving the collection advanced putrefaction, when no blood specimen is available,
of samples from either the mother (e.g. urine or hair) or the with partial or complete equilibrium of XB concentrations
newborn (e.g. urine, meconium, hair, amniotic fluid) (Gray within the corpse (Sims etal. 1999).
and Huestis 2007). There is also little information on XB concentrations in
pericardial fluid in spite of its sufficient amounts to be usable
Excised skin from injection sites (Gibson and Segal 1978). Pericardial fluid is an ultrafiltrate
Suspect injection sites could be excised and submitted for of plasma with a very similar amount of proteins, which is
analysis, to support evidence of that route of administra- contained within a tight compartment (pericardic sac) so
tion. Skin, subcutaneous adipose tissue, and skeletal muscle that it is free of contamination by microorganisms. The usual
should be sampled. The simple qualitative detection or even volume currently taken at the time of the autopsy ranges
quantitative measurement of a XB in the excised tissue only from 520mL. Moriya and Hashimoto (1999b) explored the
evidences that the XB exposure occurred and not that it was usefulness of pericardial fluid as a specimen for toxicologi-
necessarily injected. Sometimes it is forgotten that most cal analyses by comparing pericardial fluid with blood. The
XBs are distributed throughout the body from any route of authors observed fairly good correlations between blood
administration, such that any piece of skin will contain some and pericardial fluid for all analyzed XBs and recommended
amount of the XB. For such measurements to be useful, a that pericardial fluid should be added to the list of routine
similar sample of the skin from another part of the body, not autopsy specimens. Pericardial fluid was sometimes better
suspected to be an injection site, must be analyzed for com- than blood when judging results, and it was suitable for quan-
parison (Flanagan etal. 2007). Only if the concentration in the titative estimations of XB concentrations, while cerebrospinal
suspect site is substantially higher than that in the reference fluid, urine, and bile were considered useful for qualitative
site, can significant conclusions be drawn (Karch 2008). Even analysis. More recently, Contreras etal. (2006; 2007) showed
then, a perfect injection may not cause persistent elevated XB the usefulness of analyzing drugs of abuse (morphine and
concentrations at the intravenous injection site, in contrast to cocaine) in pericardial fluid.
an intramuscular or subcutaneous site.
Bone marrow
Pleural effusion and pericardial fluid There is a scarcity of research on the analysis for XBs in
Several body fluids are available at autopsy, which may be human bone marrow samples (Kojima etal. 1986; McIntyre
used as an alternative specimen for XB detection, especially etal. 2000; Raikos etal. 2001; Lafreniere and Watterson 2010).
when other fluids are absent or are of reduced quality due to The first report occurred in 1978, when amitriptyline was
post-mortem processes. identified in skeletonized human remains (Noguchi et al.
In addition, post-mortem blood XBs concentrations 1978). Since then, most experiments have been undertaken
have been shown to vary depending on the sampling site. using rabbit femoral bone marrow, which has shown linear
Therefore, although there are cases where blood is enough relationships between bone marrow and peri-mortem blood
for analytic purposes, it may not be suitable for the resolution XB concentrations for up to 24 hours for many XBs including
of the toxicological problems raised. In the laboratory, liquid tricyclic antidepressants, barbiturates, benzodiazepines, and
specimens are easier to sample than tissue, and toxicology ethanol (Winek and Jones 1980; Winek and Esposito 1981;
protocols designed for blood samples are easily adapted. Winek and Luhanik 1981; Winek etal. 1981; 1982; 1983; 1985;
Pleural effusion is a blood-stained fluid of the pleural cavity 1990; 1993; Winek and Janssen 1982; Winek and Susa 1982;
derived from liquefaction of surrounding tissues due to putre- McIntyre etal. 2000; Raikos etal. 2001; Schloegl etal. 2006b;
faction. This putrefactive effusion forms in the pleural cavity Guillot etal. 2007; Kugelberg and Jones 2007).
and increases in volume as the blood in the vascular system Bone marrow comprises vascular tissue present in the
disappears in the period of 12 weeks after death. It presents central cavities of bones (Jenkins 2008). There are two types
as a collection of dark, blood-stained and watery fluid. Thirty of marrow: red and yellow, and these can differ in composi-
milliliters of the pleural effusion should be collected from the tion in different bones. Yellow marrow consists of a basis of
connective tissue supporting numerous blood vessels and easily accessible in routine autopsies, without changing the
inert adipose (fatty) cells (Jenkins 2008). The red marrow structure of the corpse in a relevant way, in contrast to bone
(or myeloid tissue) consists of a connective tissue (stroma) marrow of vertebrae or long bones. Schloegl etal. (2006b)
that supports clusters of hemapoietic cells, white blood cells, collected bone marrow tissue from the second and third
macrophages, and a rich vascular supply. Long bones receive rib on the right side. Routinely, in every autopsy the thorax
a rich local blood supply, whereas short, flat, and irregular is opened by cutting the ribs a few centimeters lateral of
bones receive a limited supply through the periosteum. the sternum in their cartilaginous part and the sternum is
Normal human hemapoietic marrow is bright red in color taken out. For the collection of rib bone marrow, the rib has
and firm in consistency. been cut ~ 5cm from its distal end, which is approximately
There are some advantages to using bone marrow as an in the medioclavicular line where it is ossified (Figure 16)
alternative tissue for analysis in Forensic Toxicology (Raikos (Schloegl et al. 2006b). By compressing the ends of the
et al. 2001). Bone marrow is often recoverable after skel- remaining rib, the dark red bone marrow can be squeezed
etonization, it is encased in bone, and it has a high degree of out of the bone.
vascularity and a lipid matrix that may act as a XB repository
for lipophilic XBs. At this stage qualitative analysis using bone Bone
marrow seems to be very useful in documenting the presence Bone in humans has four shapes, namely long, short, flat, and
of a XB that was not expected to be present. This acquires irregular. Long bones have a central shaft, containing bone
particular importance for XB detection in cases of advanced marrow, with expanded ends. Bones exhibit two types of tis-
putrefaction, fragmentation, skeletonization, and exsanguin- sue, solid dense compact bone and spongy bone, comprising
ated human remains, and is useful when contamination of a network of bony rods interspersed with spaces containing
blood specimens is suspected in trauma cases. Nevertheless, bone marrow. Both types of tissue are found in many bones,
although putrefaction is delayed in bone marrow, usually for example, in the humerus, compact bone forms the shaft
this specimen is not routinely considered as an alternative and spongy bone is found in the ends or epiphyses (McGrath
specimen in post-mortem toxicology, unless other specimens and Jenkins 2009).
are unavailable. This can be explained mainly by the fact In cases of extensive decomposition, burnt, exsanguina-
that, during putrefaction, bone marrow is transformed from tions, or body fragmentation, only a few tissues may be avail-
spongy red marrow to a brown viscous liquid or paste-like able for examination. In these circumstances, bone (or teeth,
substance compromising any interpretation from the quan- which are one of the best preserved tissues) may be a useful
titative point-of-view (Winek etal. 1983). specimen for potentially determining the role of XBs in medi-
Until now, no specifications exist concerning which colegal cases, but only as indicative of exposure. The reports
bone should be used to obtain marrow. Bone marrow of that have been published thus far have consisted largely of
ribs has been chosen (Schloegl etal. 2006b) due to being individual case reports, with little indication of consistency
A B
C D
Figure 16. Obtaining bone marrow from the second and third rib on the right side. For collection, the ribs are cut ~ 5cm far from its distal end, which
is approximately in the medioclavicular line where it is ossified (a and b; dashed line). By compressing the ends of the remaining rib, the dark red bone
marrow can be squeezed out of the bone and aspirated (c and d).
in the particular bone tissue sampled. There are no data sug- compounds may be transferred from blood to bone to a lesser
gesting that one anatomic region is better than another, but extent. Some XBs, maybe reflecting exposure chronicity, were
long bones such as the femur are easier to work with than detected in bone, but not the corresponding blood. Similar
smaller bones. The flat bone, iliac crest (Horak and Jenkins to previous findings obtained for some XBs (McIntyre etal.
2005), irregular bone, vertebrae, long bone, femur (McIntyre 2000) there appeared to be no relationship between a blood
etal. 2000), and flat bones, sternum, and ribs (Benko 1985) XB concentration and the likelihood of a positive finding in
have been selected. Bones should be cut into small segments bone, and high blood concentrations did not always result in
(e.g. femur rings) or crushed (Drummer and Gerostamoulos elevated concentrations in bone. In a pilot study, morphine
2002). and codeine were detected in human teeth from individu-
Most XBs will be taken up by bone tissue and, therefore, als that had died of heroin overdose (Cattaneo etal. 2003).
unless volatile, will be detectable in skeletonized remains However, as for other bones, the rate and degree of entry into
(Drummer and Gerostamoulos 2002). The degree of contact the various structures of teeth (dentine and enamel) is not
of XBs to the bone structures depends on the anatomical known and likely to be slower than for long bones due to the
location of the bone and the local blood supply. Long bones lesser degree of vascularization.
receive higher blood supply comparatively to the short, flat, Further studies are required to determine the dose, blood
or irregular bones, which receive more superficial supply concentration, and frequency of XB use necessary for deposi-
through the periosteum. This intrinsic property advocates the tion in bone (McGrath and Jenkins 2009). Controlled studies
use of long bones (i.e. femur) instead of others. For certain investigating drug disposition in skeletal tissues are more eas-
XBs the interpretation of concentrations is relatively easy ily done using animal tissues as opposed to human autopsy
since either the normal or reference values are well estab- tissues. However, the direct applicability of such studies to the
lished (e.g. heavy metals), or the XB should not be present in interpretation of forensic analyses is still limited.
any concentration (e.g. strychnine), while, for others, such as
pharmaceutical drugs or drugs of abuse, the interpretation is Entomological specimens (entomotoxicology)
problematic because of limited reference levels (McGrath and When decomposition prevents traditional specimens, such as
Jenkins 2009). In addition, it should be recognized that bone blood, urine, or solid tissues, from being obtained, homog-
is continuously remodeled. Therefore, XBs incorporated in enized fly larvae (maggots), usually of Calliphoridae (blow-
bone tissue over time will be liberated and re-delivered to the fly), Sarcophagidae (flesh flies), and Muscidae (house flies)
blood, which means that a negative detection in bone does families have proved to be useful alternative specimens in
not rule out an exposure, and a positive detection will not give which XBs may be identified (Byrd and Castner 2001). They
very much information about time for exposure. Indeed, XBs are highly motile, strong-flying insects, and are typically the
distributed to bone will almost certainly remain here longer first to reach the dead body, often within minutes of death
than in blood due to the relatively slow turnover of the bone, (Byrd and Castner 2001). Depending on temperature, larvae
increasing therefore the time frame for detection. may be present as soon as 1 to 2 days after death (Byrd and
Comprehensive data on post-mortem XB findings in bone Butler 1996; 1997; 1998). XB concentrations in larvae have
were provided by McIntyre etal. (2000). Tricyclic antidepres- been found to depend on the tissue the larvae had fed on as
sants, as well as non-tricyclic drugs such as mianserin and well as on their stage of development, which is influenced by
moclobemide, antipsychotics (chlorpromazine, thioridazine, the type of XB present in the corpse (Kintz etal. 1990b; Wilson
and clozapine), benzodiazepines (diazepam, oxazepam, and etal. 1993). Indeed, various studies point to the influence of
temazepam), and major corresponding metabolites, were XBs in the development of larvae, which can compromise an
identified in sections from human femoral bone and bone accurate estimation (either by excess or deficit) of the post-
marrow. The majority of targeted XBs generally matched mortem interval (Goff etal. 1991; Arnaldos etal. 2005; George
qualitatively to the blood results. The concentrations of pri- etal. 2009).
mary metabolites were lower compared to those of the parent It appears to be critical that larvae collected for XB analysis
XB. In a rabbit model, methamphetamine was always detect- from a decomposed body are frozen and analyzed as soon as
able in bones that were kept under various conditions over a possible after collection, since larvae rapidly eliminate XBs
2-year period (Nagata etal. 1990). Elevated aluminum levels when removed from a food source (Karch 2008). Nevertheless,
in liver, brain, and bone specimens helped to diagnose the even under refrigerated conditions, when larvae are in a state
most probable cause of death in dialyzed patients (de Wolff of diapause, slow bioelimination of XBs still occurs over the
etal. 2002; Cengiz etal. 2006). Some important conclusions course of several weeks (Sadler et al. 1995). The choice of
were recently ascertained by McGrath and Jenkins (2009). The where larvae are best collected from the body needs further
authors observed that several factors influence the deposition study (Karch 2008). Interpretation of positive findings seem
of XBs in this matrix, including acute vs chronic exposure, to be most useful if the larvae are collected at the site of their
distribution at the time of death, site of bone collection and food source, such as any remaining muscle or liver, under the
bone type, and physicochemical characteristics of the XB. premise that XBs detected in fly larvae feeding on a body can
XBs with short half-lives or those inherently unstable, such as only have originated from the tissues of that body (Pounder
6-acetylmorphine and olanzapine, may not be stored in bone. 1991; Pounder et al. 1996a). This assumption seems to be
In addition, polar metabolites and highly protein-bound supported by earlier studies where a quantitative relationship
was suggested between the morphine concentrations in the c. What was the contribution of alcohol or other XBs for a
larvae and the livers on which they fed (Introna etal. 1990; person to fall down a flight of stairs?
2001). By contrast, other studies suggest that the analysis of d. In the case of a death in a fire, was the victim burned?
fly larvae provides only qualitative data (Kintz etal. 1990a; Is the death consistent with smoke inhalation? Was the
b; c). However, in these studies, larvae specimens were victim alive or already dead when the fire occurred
obtained without taking into account that larvae XB concen- (Dinis-Oliveira et al. 2010)?
trations are based on the tissue on which they fed (Pounder
e. Were drugs of abuse used to incapacitate a girl during
1991; Pounder etal. 1996a), since specimens were collected
an alleged date rape?
from multiple sites and they were pooled before analysis.
Nevertheless, at this stage, due to the wide variations dem- f. Was a XB used to commit suicide?
onstrated in the various studies cited above, it seems more g. Was a person murdered with a XB?
reasonable to consider that accumulation of XBs in larvae
h. Is the concentration of the XB able to explain the toxic
is unpredictable and quantification unreliable (Sadler etal.

symptoms or the fatality?
1995). Even if used only for qualitative analysis, larvae toxi-
cological analysis could play an important part in detecting i. What was the route of XB exposure?
XBs and can contribute to establishing the cause, the manner, j. To comment on acute or chronic intake/exposure;
and the mechanism of death. k. To classify the concentrations as therapeutic or toxic;
To avoid environmental (surface) contamination, a source or
of interpretive error, larvae should be washed with deionized
water prior to analysis (Gagliano-Candela and Aventaggiato l. To roughly estimate the time interval between exposure/
2001). For analysis, larvae are homogenized and processed intake and death.
in the same manner as human tissues. XBs extraction tech-
The forensic toxicologist must be able to provide accurate and
niques from chitinized insects are similar to those performed
concise answers to these queries using language that is eas-
on hair samples. The first reported use of fly larvae in XBs
ily understood by lawyers, jurors, and judges. Furthermore,
analysis occurred as recently as 1980 and involved a pheno-
the opinions expressed by the toxicologist must always be
barbital case (Beyer etal. 1980). Since then numerous XBs

impartial and based only on the scientific facts involved.
have been identified in fly larvae including benzodiazepines,
Comprehensive information already prior to sampling is an
tricyclic antidepressants, opiates, cocaine, and the organo-
important precondition that even allows answering such
phosphate, malathion (Gunatilake and Goff 1989; Kintz etal.
complex questions and supporting reliable interpretation
1990a; b; c; 1994; Nolte etal. 1992). Gagliano-Candela and
of the analytical results obtained from ante-mortem or post-
Aventaggiato (2001) published an interesting review on the
mortem material.
XBs detected in entomological specimens and on experi-
A standard report format is very helpful and results should
mental studies.
be reported in appropriate units, preferably written out in full
to avoid confusion (e.g. between mU/L and U/L), indicat-
Reporting results and interpretation aspects ing the measurement uncertainty if desirable, with appro-
priate reference data or information to assist the requester
One of the major concerns that must be always present is that in understanding their significance (Jickells and Negrusz
the results of a toxicological investigation may be included 2008). This may, for example, be a reference to legislation,
in court testimony. In addition, as an expert in the field of production criteria, and published papers in the scientific
intoxications, the forensic toxicologist may be asked to give literature, or the expected values for clinical investigations
an opinion either by a report or in the courtroom. Often, the (Jickells and Negrusz 2008). Special care is required when
defense will call its own experts to dispute the opinions given comparing results, especially when it is needed to choose
by the plaintiff or prosecutors toxicologist. Cases involving the correct relative molecular mass (Mr, molecular weight)
XBs, for several times become a debate of experts and such if the XB is supplied as a salt, hydrate, etc., since this can
proceedings can be quite intense, with the jury left to decide cause great discrepancies, especially if the contribution of
which expert opinion seems most likely to be true. In assess- the accompanying anion or cation is high (Flanagan and
ing the evidence of the analytical toxicological results, the Connally 2005). Analytical measurements should be reported
courts are concerned especially with the experience of the in terms of the free acid or base and not the salt. Care is also
analyst, the ability to prove continuous and proper chain of needed in comparing blood (e.g. mg/L) and tissue (e.g. mg/
custody compliance, and the analytical methods used. The Kg) concentrations: 1L of water has a mass of 1 Kg at 4C,
following are some examples of the questions or comments but slightly variations occur at different temperatures. The
that the forensic toxicologist might be asked by medical, legal, debate over choice of units has been reviewed by Flanagan
or law enforcement professionals (Skopp 2004; Stripp 2006): (1998; 2004). An example of a Forensic Toxicology report is
given in Figure17.
a. Was the driver intoxicated at the time of the accident? It is important to have always the conscience that there
b. Was XB-induced psychosis a likely explanation for a are no absolute rules for the interpretation of toxicology
persons bizarre and violent behavior? results since it is not a simple matter, as becomes obvious
by the revisions of literature performed by several authors to site within the same cadaver. The extraction efficiency
(Levine et al. 1990; Flanagan 1998; 2004; Drummer and of the XB, metabolite or internal standard from outdated
Gerostamoulos 2002; Drummer 2004; 2007a; Flanagan and blood bank blood may sometimes be markedly different
Connally 2005; Kugelberg and Jones 2007). The more infor- from decomposed case blood.
mation that is available to, and considered by, the interpreter,
the more likely are the conclusions reached to be accurate. Although it is practically impossible to know the absolute
In the courtroom, lawyers, jurors, and judges often view all or true concentration of XB in a specimen, the degree of
science, including the forensic sub-specialties, in absolute confidence increases with the specificity of the analysis,
terms. Certainly, if the toxicologist does his or her job prop- with replication, or in some cases by applying multiple ana-
erly, the laboratory findings will have the required accuracy lytical methods of different physical or chemical principles.
(Karch 2008). It is also obvious that the interpretation of any The use of GC-MS with multiple ion monitoring and stable
toxicological results will be no more reliable than the analyti- isotope (e.g. deuterated) labeled internal standards will usu-
cal result itself. All interpreters must know the limitations of ally provide a higher degree of confidence in the accuracy of
the testing and the following questions should be taking into the analytical result than using an immunoassay procedure
account (Karch 2008): (Karch 2008). The completeness of the analysis should also be
considered. It is impossible to test for every single XB during
a. Was the standard material used to prepare the calibra- routine screening tests. However, a careful review of the medi-
tors pure and correctly identified? cations or other potential XB available to the victim should
assist the laboratory in focusing the toxicological analysis.
b. Was the salt or water of crystallization properly taken
Attempts to interpret toxicology findings solely on the
into account?
basis of so-called normal or reference ranges are irresponsi-
c. Was the calibration properly prepared and validated in ble. More erroneous becomes the interpretation in the field
the range where the specimens were measured? of post-mortem Forensic Toxicology. Probably, there is no
d. Was the assay adequately verified by quality control forensic toxicologist or pathologist alive who has not used
samples? published tables as a reference when trying to interpret post-
mortem blood concentrations. Tables of such values became a

e. Was the assay sufficiently specific?
necessary evil due to the sheer volume of medical and foren-
f. Could other XBs or metabolites have interfered with sic literature. However, they unfortunately perpetuate the
analysis of the specimen, either by obscuring the target myth that post-mortem toxicology results can be interpreted
analytes or by increasing the apparent concentration? solely using, or heavily relying on, so-called therapeutic,
g. If the specimen was analyzed only once, what was the toxic, and fatal ranges (Karch 2008). Although tables of XB
potential for accidental contamination? concentrations can serve as a useful reference point, it should
h. Was there a matrix effect? be borne in mind that many of the values in these tables
are derived mostly from serum or plasma data from living
i. Was the recovery of the XB from the specimen the same, patients (Schulz and Schmoldt 1994; 1997; 2003; Winek etal.
relatively, as from the calibrators? 2001), that the ranges are seldom referenced to published
j. Even using similar matrix calibrators (e.g. whole blood) cases, and that they are not take into account or state other
is not necessarily a guarantee of the correction of matrix variables such as post-mortem redistribution (also referred to
effects when using post-mortem blood, since, by its as the toxicological nightmare (Pounder and Jones 1990)),
nature, it is variable from case to case, or even from site diffusion and metabolism, tolerance, time of survival after
TOXICOLOGICAL REPORT N2010/_____

Victim name:________________________________________________________________
________________________________________________________________
Requested by:
Toxicological Analytical Sample (refer the Resultand units Forensic analyst
parameter technique and anatomic place of signature
limit of detection blood collection)
(LOD)
Commentary:_______________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
Date, place and signature of the service director:__________________________________

_________________________________________________________________________
Figure 17. An example of a Forensic Toxicology report.

intoxication, or the presence of other XB (interactions), natu- fluid, through the capillaries and into the larger blood ves-
ral disease, or injury. In addition, specific references to case sels of those organs, resulting in higher concentrations of the
data and further information are lacking in most instances. XBs in surrounding tissues than true concentrations at the
For example, interpretation of opioids concentrations may be time of death. Once cells die, their membrane integrity is lost,
very dependent on how long the person has been exposed to and the cells leak their contents (XBs, electrolytes, etc.) into
the XB and at what dosage. The inappropriate use of tables the extracellular space. In these cases, post-mortem levels in
can result in over- and under-estimation of the potential tox- central (cardiac) blood can be several times higher than they
icity of a XB depending on the degree of tolerance developed, were ante-mortem, due to passive diffusion from myocardial
natural disease, route of administration, and whether other cells into blood in the cardiac chambers or diffusion from
XBs are present. In these cases setting lethal post-mortem adjacent lung tissue into the great vessels in the thorax (aorta,
levels is very difficult, since the range of post-mortem over- inferior vena cava, etc.) (Yarema and Becker 2005; Holland
dose levels often overlap with those seen in patients on 2009). Post-mortem diffusion generally refers to the diffusion
chronic opioid treatment. Post-mortem concentrations have of XBs along a concentration gradient, from an area of high
been over-interpreted in the past, and good evidence should concentration to an area of low concentration located nearby.
be required before lethal concentrations are defined in The usual scenario is when a high concentration of XB in the
the future. The same rationale is obvious when analyzing stomach contents (e.g. after an overdose) causes elevated
literature data concerning ethanol. The lethal concentration concentrations of the XB in nearby tissue (e.g. proximal lobe
of ethanol has sometimes been reported as above 500 mil- of the liver) or blood. For example, in life, ethanol is absorbed
ligrams per 100 milliliters but a patient was still talking when from the small intestine rather from the stomach. However,
her serum ethanol concentration was > 1500mg per 100ml, after death the stomach wall becomes permeable to ethanol,
three times this fatal value (Johnson etal. 1982). Therefore, which then diffuses into adjacent tissue and blood vessels
while these tables may be of some (and not all) use in clinical (Pounder and Smith 1995; Pounder etal. 1996b). Such diffu-
toxicology, they are of very limited value for the interpretation sion through previously impermeable barriers may be most
of post-mortem toxicology results and can be very misleading. important for small non-polar molecules (Pelissier-Alicot
Nevertheless, some important compilations providing refer- etal. 2003). Much is still unknown about the extent to which
ence levels on post-mortem femoral blood samples have been post-mortem changes in XB concentration occur, and the dif-
published (Druid and Holmgren 1997; 1998; Reis etal. 2007). ferent XBs affected. The likelihood that post-mortem samples
Such data are important and additional compilations using of blood reflect ante-mortem XB levels will depend on the
this approach are obviously encouraged. circumstances of the death and post-mortem sampling, as
Kinetics is an invaluable tool to help understand the well as whether post-mortem redistribution is likely to take
time course of XB in the body. In the living, it can be used place. The characteristics of the XB itself, which will influence
to determine duration of action, inter-individual differ- the likelihood of post-mortem redistribution, are the volume
ences in peak plasma concentrations and clearance, and of distribution, the pKa, and the lipophilicity (Pounder and
the likely effectiveness of different pharmaceutical formula- Jones 1990; Pelissier-Alicot et al. 2003; Yarema and Becker
tions. However, rarely can kinetics be applied successfully to 2005; Holland 2009). If the volume of distribution of the XB is
post-mortem toxicology. The kinetics of all XBs in the body is high, ie a volume of distribution > 1L/Kg, then that XB is more
characterized by absorption, distribution, metabolism, and distributed to tissues, and post-mortem redistribution is more
excretion. All these steps affect the concentrations of XBs and likely to occur. The pKa of the XB is defined as the pH at which
therefore the interpretation of ante-mortem and post-mortem the XB is 50% ionized. A basic XB, i.e. pKa > 7, will exhibit
analytical toxicology results. Nevertheless other issues must more post-mortem redistribution. This can be explained by
be considered when interpreting post-mortem toxicological two mechanisms: (i) basic XBs are more prone to concen-
results, since the human body is not a static entity after death. trate in tissues before death, and (ii) the contents of a cell are
Post-mortem redistribution represents a special concern that largely aqueous and become acidic after death. Since a basic
misled the interpretation in post-mortem Forensic Toxicology. XB will be progressively more ionized in an increasingly acidic
Toxicologists participating in forensic cases involving XBs medium, after cell lysis occurs, basic XBs will distribute more
likely to undergo post-mortem redistribution must be aware readily as a result of being transported in the acidic fluid in
of its potential contribution to the post-mortem XB concen- which they are dissolved. Highly lipophilic XBs are also more
tration. In addition, often no ante-mortem or peri-mortem distributed in tissues, and are therefore more likely to exhibit
XB blood is available for analysis, and clinical information post-mortem redistribution. A comprehensive discussion of
about the deceased at the time of death may not exist if no the possible mechanisms for post-mortem redistribution and
medical attention was provided. Post-mortem redistribu- the XBs affected was published by Pelissier-Alicot etal. (2003)
tion reflects the diffusion and redistribution of XBs occur- and Yarema and Becker (2005).
ring in the body in the interval between death and autopsy Another topic that must be considered when interpret-
sampling (Cook etal. 2000) along a concentration gradient. ing XB concentrations is the sum, or even more complex,
Redistribution generally refers to the release of XBs from the synergy of the effects of all of the XB detected. In the
areas of higher concentration (e.g. from binding sites in cells majority of cases in clinical and Forensic Toxicology, more
or tissues) and subsequent passive diffusion into interstitial than one XB is involved. Multidrug therapy and abuse is
prevalent, and this, together with the added problems of and dichlorodiphenyltrichloroethane (DDT) are regularly
self-medication with over-the-counter drugs and the wide- ingested from food sources or inhaled as environmental
spread use of alcohol, makes interpretation of data even contaminants and retained by the human body, but in
more complicated. Several deaths often involve multiple amounts that are insufficient to cause obvious deleterious
drugs of the same type (e.g. benzodiazepines or narcotics), effects (Klaassen 2008). Ethanol represents another typical
individually present in therapeutic amounts, and often example, since it is produced after death (Kugelberg and
in combination with alcohol. Interpretation of blood XB Jones 2007). Conversely, blood XB concentrations that
concentrations in these cases has to take into account any exceed therapeutic concentrations by 1020-times are
disease that may be present, and the total amounts of XBs consistent with intoxication or death, excluding an obvious
and alcohol. XB interactions can be divided into two types: contamination problem. In addition, the higher the parent
pharmacokinetics and pharmacodynamics. Both divisions XB-to-metabolite ratio, the more likely an acute intoxica-
can originate addition, synergism, potentiation, and antago- tion has occurred (Karch 2008). Finally the possibility of
nism interactions. endobiotics involvement in the intoxication should not be

Artifacts of absorption and distribution must also be disregarded (Lam etal. 2006; Chambellan-Tison etal. 2007).
recognized when interpreting post-mortem blood concen- A compound apparently innocuous as pure water will, if
trations. For example, it is quite common to find grossly ingested in sufficient quantity (as occurs in schizophrenic
elevated concentrations of lidocaine in cases where resus- patients suffering from polydipsia), can cause incapacitating
citation has been unsuccessfully attempted (Moriya and electrolyte imbalance, hyponatremia, confusion, rhabdomy-
Hashimoto 1997c). Forensic toxicologists should be always olysis, systemic seizure, and edema or even death (Vieweg
aware of such a phenomenon. Concentrations may be 25- etal. 1985; Chen and Huang 1995; Arieff and Kronlund 1999;
times those normally considered therapeutic when lido- Hayashi etal. 2005).
caine is given by intravenous infusion for the treatment of
cardiac arrhythmias. If lidocaine is administered as a bolus
intracardiac injection and normal cardiac rhythm is never
Concluding remarks
established, very high local concentrations will result in the One of the main functions of the forensic toxicologist is to
cardiac blood. These could be interpreted as fatal unless produce results and to interpret them in order to be used in
all the circumstances are considered. In addition, jelly lido- criminal prosecutions, which implies rigor to survive court
caine formulations are usually used at endotracheal intu- cross-examination and medicolegal scrutiny (Drummer
bation in emergency medicine, being frequently detected 2007b). As a general view, probably extended to all branches
in blood of cadavers that had received cardiopulmonary of the forensic sciences, much of the information transmitted
resuscitation (Moriya and Hashimoto 1997c; 1998a; Pounder in Forensic Toxicology has been obtained from case stud-
1997). Devices that automatically deliver medication by the ies described at meetings or in published works (Drummer
parenteral route (such as transdermal patches) can lead 2007a). Interpretation has therefore been empirical, with
to artificially extremely high post-mortem local blood con- typical intoxication data found in the literature serving
centrations (misinterpreted as an overdose) unless they are as benchmarks (Fanton et al. 2009). Collectively this has
switched off or disconnected quickly (Anderson and Muto formed much of the basis of knowledge in this discipline,
2000; Solarino etal. 2010). Since these patches rely primarily which led to criticisms from some groups, of other medi-
on passive diffusion across a rate-limiting membrane for XB cal disciplines in medical faculties, regarding the lack of
delivery, the concentration of the analyte in the local area clear evidence of some conclusions that are drawn in spe-
will continue to rise after death, albeit at a slower rate. Since cific cases and the bias that can lead to poor quality and
blood circulation through the skin obviously stops after quantity of research (Drummer 2004; Guzelian etal. 2005).
death, the XB will no longer be transported away, except Indeed, Forensic Toxicology has different aims and relevant
by diffusion, allowing a local build-up of XB (Jickells and intersections with arts and socio-scientific disciplines (such
Negrusz 2008; Karch 2008). as law) in comparison with Clinical Toxicology, a fact that
Despite these concerns, some aspects of interpreta- increases the number of variables to be considered (Madea
tion remain relatively straightforward. A negative result etal. 2007). In addition, controlled randomized, prospec-
is not sufficient to establish that the XB is not implicated tive cross-sectional, cohort, or case-control studies can only
in the intoxication case. Negative results are sometimes rarely be applied in Forensic Toxicology. Specifying the opti-
assumed because standard screening tests have shown mum size of a sample in order to attribute credibility to the
no suspicious results or the XB disappears before analy- results is also always problematic in Forensic Toxicology.
sis. Additionally, a negative result below a defined limit of For many laboratories, sample sizes of fewer than 10 are
detection can be interpreted as lack of acute exposure to common, but when investigations of cases are conducted
that analyte or non-compliance in the case of therapeutic with the view to compare them and illustrate common fea-
drugs (Karch 2008). A positive qualitative analysis never tures, sample sizes will need to be much higher. For exam-
solely provides conclusions of the involvement of an XB in ple, understanding the association between illicit drug use
the intoxication, but is only one of many pieces of evidence. and crash risk has required samples sizes of many hundreds
For example, minute quantities of cyanide, arsenic, lead, to thousands (Mura etal. 2003; 2006). In the literature, there
are many studies that have provided significant knowledge Declaration of interest
regarding the specimens and amount to be collected. Some
of these examples were discussed and harmonized guid- Ricardo Dinis-Oliveira acknowledges FCT for his Post-Doc
ance is attentively given in this paper. It is recommended grant (SFRH/BPD/36865/2007). The authors report no con-
that medical examiners/pathologists, and if necessary the flicts of interest. The authors alone are responsible for the
police investigators or judicial authority, discuss the case content and writing of the paper.
with toxicologists in advance of collection to ensure that
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Toxicology Mechanisms and Methods, 2010; 20(7): 363414

Collection of biological samples in forensic toxicology

(Received 09 April 2010; revised 12 May 2010; accepted 16 May 2010)

abortion). tive operation of a motor vehicle, as well as doping in

Institutional case number identifier or request

Cardiac Peripheral Other

Date and collection time:

intented to analyze. The principles outlined by Bush etal. etal. 2007).

Using capital letter, fill the request in a

Forensic laboratory logotype

REQUEST Ante-mortem Post-mortem

Address for report:_____________________________________ Judicial proceeding number:________________

Male Female Weight:__________ Height:____________ Occupation (details of end-product of factory or

Yes No If yes, give details:_________________________________________________________________

Are the analysis urgent? Yes No Telephone:____________

Occupational accident Unknown Other:_______________________________________________________

Intoxication suspicion: Yes No If no, give brief details:___________________________________________

Ethyl alcohol Carbon monoxide Drugs of abuse* Medicines* Pesticides* Other*

Yes No If yes, give details of the results:______________________________________________________

Yes No If yes, give details:_________________________________________________________________

Figure 3. continued on next page

Relevant symptoms: Diarrhea Vomiting Thirst Blindness Constipation Cyanosis Jaundice

Loss of weight Shivering Convulsions Miosis Mydriasis Delirium Coma Sweating

Affected organs , which:________________________ Others: Give details:__________________________

Forensic laboratory logotype

Age:__________ Male Female

N of the specimen handled: N of the specimen handled:

N of the specimen handled: N of the specimen handled:

-Collected in traumatic cases;

-10mL for plastic universal container with screw cap;

Table 1. continued on next page

-Useful to guide blood analysis;

Table 1. continued on next page

Table 1. continued on next page

will contain a higher concentration of XBs compared with a Urine

Figure 7. To obtain urine, for victims not subjected to internal examina-

be empty, it is important to aspirate as much urine as pos-

ebrospinal fluid for XB concentrations;

concentrations to those in vitreous humor;

studies comparing vitreous humor and blood ethanol

lost and the pH increases. Dawes and Jenkins (1964) dem-

Lopez etal. 2007; Gallardo and Queiroz 2008).

with blood. Nevertheless, XBs concentrations in the lobe Lung

Figure 14. For skeletal muscle, normally, specimens of iliopsoas mus-

mortis are essentially the same as those without it (Levisky

1993a; b; Kintz 2004; 2007; Kintz etal. 2007a). In drug-facil-

is unpredictable and quantification unreliable (Sadler etal.

barbital case (Beyer etal. 1980). Since then numerous XBs

mortem blood concentrations. Tables of such values became a

TOXICOLOGICAL REPORT N2010/_____

Date, place and signature of the service director:__________________________________

Figure 17. An example of a Forensic Toxicology report.

nism interactions. endobiotics involvement in the intoxication should not be

text. Anal Bioanal Chem 390:11591166.

Sci 36:537542. and benzoylecgonine in urine: a review. J Anal Toxicol 18:104109.

81:460463. case analysis. Pediatr Pathol 12:463468.

a review. J Anal Toxicol 16:19. York: Springer. pp 141177.

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Address for report:_____________________ Judicial proceeding number:

Male Female Weight: Height:__ Occupation (details of end-product of factory or

Affected organs , which: Others: Give details:__