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Toxicology Mechanisms and Methods Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/itxm20
Collection of biological samples in forensic toxicology c
d
c
R. J. Dinis-Oliveira, F. Carvalho , J. A. Duarte , F. Remião , A. Marques, A. Santos & T. Magalhães a
Faculty of Medicine, University of Porto, Porto, Portugal
b
Department of Clinical Analysis and Public Health, Center of Research in Health Technologies (CITS)-IPSN-CESPU, CRL, Vila Nova de Famalicão, Portugal c
REQUIMTE, Department of Toxicology, Faculty of Pharmacy, University of Porto, Porto, Portugal d
CIAFEL, Faculty of Sport, University of Porto, Porto, Portugal
e
North Branch, National Institute of Legal Medicine, I.P.
f
Center of Forensic Sciences, Portuguese Science and Technology Foundation, Lisboa, Portugal g
Biomedical Sciences Institute ‘Abel Salazar’, University of Porto, Porto, Portugal Published online: 17 Apr 2015.
To cite this article: R. J. Dinis-Oliveira, F. Carvalho, J. A. Duarte, F. Remião, A. Marques, A. Santos & T. Magalhães (2010) Collection of biological samples in forensic toxicology, Toxicology Mechanisms and Methods, 20:7, 363-414 To link to this article: http://dx.doi.org/10.3109/15376516.2010.497976
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Toxicology Mechanisms and Methods, 2010; 20(7): 363–414
REVIEW ARTICLE
Collection of biological samples in forensic toxicology R. J. Dinis-Oliveira1,2,3, F. Carvalho3, J. A. Duarte4, F. Remião3, A. Marques5,6, A. Santos1,5,6, and T. Magalhães1,5,6,7 Faculty of Medicine, University of Porto, Porto, Portugal, 2Department of Clinical Analysis and Public Health, Center of Research in Health Technologies (CITS)-IPSN-CESPU, CRL, Vila Nova de Famalicão, 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 Downloaded by [46.50.82.114] at 13:12 14 August 2015
1
Abstract Forensic toxicology is the study and practice of the application of toxicology to the purposes of the law. The relevance 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 special 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 should facilitate the assessment of sample quality and the analytical result obtained from that sample. The knowledge 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 collected 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; storage and preservation; interpretation.
Introduction to forensic toxicology Forensic science represents the application of the different scientific fields to the legal system proceedings. Forensic toxicology results from a hybridization of modern Analytical Chemistry and Fundamental Toxicology, and their application to the purposes of the law, in order to elucidate questions that occur in judicial proceedings related to intoxications. Forensic Toxicology is concerned primarily with the medicolegal aspects of the harmful effects of xenobiotics (XBs) on humans and animals (Langman and Kapur 2006). However, the analysis and identification of medicines and the maintenance of agricultural, industrial, and public health legislation (to ensure clean air, pure water, and safe food supplies) are also fields of Forensic Toxicology, although associated with civil
courts rather than criminal courts. In any case, a good forensic toxicologist must be prepared to answer several questions: The traditional one that must be answered is ‘Has this person been poisoned?’ Supplementary queries follow if the result is positive, such as ‘What is the identity of the poison?’, ‘How was it administered?’, ‘What are its effects?’, and ‘Was it a dangerous or lethal amount?’ In other words, the forensic toxicologist should perform qualitative and quantitative analysis and must interpret the probable role that a XB (or sometimes a endobiotic) played in the case (Jickells and Negrusz 2008). According to the American Chemical Society, there are ~ 21 million registered compounds, and the European Commission estimated that ~ 100,000 chemicals were in use (Greim and Snyder 2008)— 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:
[email protected] &
[email protected] (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
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364 R. J. Dinis-Oliveira et al. In the field of Forensic Toxicology, poisoning (or intoxication) can be defined as: ‘An individual’s medical or social unacceptable condition as a consequence of being under influence of XBs in a dose too high for the concerned person’ (Uges 2001). For medical and legal purposes, it is important to know how the victim became poisoned. All medications can have unwanted but unavoidable side-effects. Doctors must judge whether the cure is better than the ailment. When a drug is deliberately or accidentally taken in such a dose that a patient’s body cannot adequately metabolize and eliminate it, poisoning may ensue. A patient with terminal cancer and unbearable pain can receive strong painkillers, such as morphine in high doses. The risk of such treatment is death of the patient, either directly or indirectly as a result of the treatment. Is this acceptable? Do reasonable alternatives exist? What would be the patient’s choices without treatment? The inclusion of the term ‘medical’ in the definition of poisoning is important, as sometimes a doctor has to exceed the maximum safe dose of a certain drug when a disease is believed to be worse for the patient than the overdose (Uges 2001). By adding the clause ‘social and/or legal’ to the definition of ‘poisoning’, it is emphasized that the concept of intoxication depends on the victim’s normal state and on prevailing cultural standards and laws. These factors strongly differ from one country to the next (e.g. euthanasia, chemical abortion). Since poisoning is considered to be a medically or socially unacceptable overdose of a XB, it is important to know how somebody could be poisoned. In general, there are three possible ways of causing poisoning (adapted from Uges 2001): a. Accidental poisoning, usually results from an accident, error, carelessness, or an unexpected situation in the working environment. House accidents with babies and children are also frequent due to their propensity to explore surroundings by putting things in their mouths. Intoxication due to medical or paramedical treatment, so-called iatrogenic intoxications, also belongs to the category of accidental poisoning. Active therapeutic drug monitoring can decrease the number of unwanted effects; however, if a doctor is charged with malpractice it is important to know whether it might be a case of poisoning or just an unavoidable outcome. In the past, iatrogenic intoxication was accepted as a calculated risk. Nowadays, patients are ‘clients’ and thus less likely to accept this risk, even resorting to civil or criminal proceedings to get compensation. Expert forensic toxicologists play a crucial role in such matters, weighing the interests of the patient with those of the physician or nurse objectively and honestly; b. Experimental poisoning, commonly results from selfmedication or experimentation (even recreational) with drugs of abuse; and c. Intentional poisoning. Someone is intoxicated on purpose, either by self-administration (as occurs in attempted suicide or to get attention because of psychological or social problems (himself Münchausen’s
syndrome)) or own request (euthanasia), or by being the unwitting victim of intoxication, as in murder (attempted homicide), in sexual assault, or as consequence of Münchausen’s syndrome by proxy (the parent or guardian of a child exaggerates or creates symptoms of illness for their child/children in order to gain investigation, treatment, attention, sympathy, and comfort from medical personnel). The field of Forensic Toxicology can be roughly divided into three distinctly separate areas: Post-mortem Forensic Toxicology and ante-mortem Human-Performance Forensic Toxicology or Forensic XB testing (Goldberger and Polettini 2002): a. In post-mortem Forensic Toxicology, the forensic toxicologist contributes to establish the cause and mode of intoxication or death through the analysis of various fluids and tissues obtained during autopsy. Some of the most notorious stories in the popular media are focused on death investigations; b. In ante-mortem Human-Performance Forensic Toxicology, the forensic toxicologist is responsible for evaluating the role of XBs in the modification of human behavior, usually applied to traffic safety and the respective operation of a motor vehicle, as well as doping in sport; and c. In ante-mortem Forensic XB testing, the forensic toxicologist is responsible for demonstrating prior use or abuse of selected XBs through the analysis of body fluids, usually urine. Results from these tests are usually applied to the workplace setting. People who work in law enforcement, many government agencies, and many private companies are required to undergo drug testing as a condition of their hiring or maintaining a job. Most cases that enter a Forensic Toxicology laboratory start with the suspicion that a XB is present in the body. The identification and quantification of a XB is crucial and it is highly dependent on continued improvements and on the development of new analytic instruments or techniques. First, the forensic toxicologist will apply a screening test to establish whether there are any XBs present. Subsequently, a qualitative or a quantitative confirmatory test is performed. Quantification of the XB is necessary to infer that the amount is compatible with fatal poisoning. Finally, interpretation is carried out. In laboratory medicine, the majority of ‘laboratory errors’ result from or originate from the pre-analytic phase and not from problems concerning the analytical process (Plebani and Carraro 1997; Witte et al. 1997). Therefore, it must be recognized that—even with technological advances—accurate, forensically defensible results are predicated on the quality and type of specimens provided, and the documentation of each specimen’s origin and history. Thus, even an analytically ‘accurate value’ may be subject to misinterpretation when the XB concentration in a single blood specimen is used to explain the circumstances surrounding
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Forensic toxicology 365 intoxication, particularly when the XB concentrations are not excessively high or low. In principle, most of the pre-analytic pre-requisites for the quality of the samples considered in clinical chemistry will also apply to the quality of ante-mortem and post-mortem specimens for Forensic Toxicology (Skopp 2004). However, toxicological investigation in ante-mortem and post-mortem Forensic Toxicology must be regarded as unique. The pre-analytic phase covers a number of conditions such as the request, the time interval between death and finding of the victim, collection of biological material, its storage and transportation, registration, security, preparation of the specimens including freezing, thawing, and aliquoting, administered therapies (Irjala and Gronroos 1998), as well as the involvement and the cooperation of various professionals such as an emergency physician, the police, the mortician, and the autopsy staff, including the forensic toxicologist (Skopp 2004). Up to date, best practice guidelines do not exist to assist coroners and pathologists when toxicological analyses are required. In the following sections, an appraisal on pre-analytic aspects and procedures in ante-mortem and post-mortem toxicology based on current published literature and our own experience is given. Labeling, collection devices, and containers The first step in the specimen collection process (including evidence collection) is to ensure that the specimen containers are labeled appropriately. It is essential that the sample and any intermediate containers used to carry it are labeled in sufficient detail to eliminate any doubt about the sample origin (Moffat et al. 2004). Without attention to this detail, all other activities that occur with the specimen(s) may become compromised. As a minimum, the label should include the following information (Figure 1): (1) institutional case number identifier or request number; (2) name of the victim or other identifier; (3) specimen type (blood, liver, kidney, etc.) and anatomic place of blood collection, when applicable (heart blood, femoral blood, etc.); (4) signature or initials of the collector; and (5) date and time of collection. Finally, self-adhesive tamper-resistant stickers should be placed over both the specimen container and transport container lids to document specimen integrity (Karch 2008). The tape seal shall bear collector’s initials and the collection date. Alternatively, all collected specimens for a given case
may be placed in a tamper-evident container labeled with the case number and name initials. This protocol is particularly useful in institutions with large number of cases where specimens may not immediately be transferred to the toxicology laboratory. A unique identification number that accompanies the sample at all stages is a valuable safeguard (Moffat et al. 2004). Samples collected for clinical purposes (or even for the coroner) are often not of ‘evidential’ quality, but such samples may be all that is available. If appropriate, DNA testing may be used to establish the origin of samples when there is concern over sample identity. Biological fluids can be collected using either wide-bore pipettes or disposable hypodermic syringes with appropriate needle gauges and lengths. For each specimen, separate disposable or clean devices and instruments such as scalpels, clips, and forceps should be used to avoid contamination. Additionally, samples must be collected in separate containers, the collector must only be working with one specimen at a time, and the autopsy staff must maintain the cleanliness of the specimen container (Karch 2008). All spillage on the outside of the container should be rinsed off and decontaminated using 10% bleach solution (Karch 2008). The choice of sample container depends on its intended purpose and should be decided by the analyst. Samples should be collected in such a way as to avoid both loss of analyte and the introduction of contaminating substances that could interfere with the analysis and interpretation of the analytical results. Usually, the best container to utilize when collecting and storing biological fluids or tissue specimens is glass in appropriate storage racks, since glass is inert and does not contain any plasticizer contaminants (Moffat et al. 2004; Jickells and Negrusz 2008; Karch 2008). Particularly, if volatile solvents (e.g. resulting from solvent abuse or intoxication with anesthetic gases) are to be analyzed, glass (with a cap lined with metal foil or Teflon-lined lids) is preferred (or mandatory), in view of the fact that greater losses may occur if plastic containers are used. It is also important to collect and seal the specimen in a container as soon as possible after opening the cadaver (Karch 2008). The tube should be as full as possible to minimize headspace and should only be opened when required for analysis and only when cold (4°C). With the exception referred for gases or volatile solvents, plastic is almost always used, since it does not break, and 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.
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366 R. J. Dinis-Oliveira et al. for collection of fluid and tissue specimens. Whether a facility chooses glass or plastic, it is important that the laboratory carefully evaluates the potential contamination of the container before routinely collecting specimens in it, especially when plastic is utilized. An example of the type of study that should be conducted is seen in that reported by Faynor and Robinson (1998). In addition, the nature and potential for contamination can be evaluated by analyzing XB-negative biological fluids stored over time in the container. Methods using chromatography or mass spectrometry are particularly susceptible to some of these interferences as these materials result in extraneous peaks, co-eluting peaks, and more subtle problems of ion suppression or enhancement (Zhang et al. 1996; Murthy 1997; Wu et al. 1997; Drake et al. 2004). Neverthless, interferences from collection tubes extend beyond chromatography-based methods to other types of methods (Yen and Hsu 2004; Bowen et al. 2005). If plastic is used, it must be carefully chosen to ensure that it does not break when frozen (McCurdy 1987). For example, polystyrene is subject to cracking under these conditions, whereas polypropylene is not. Plastic tubes containing separator gels could constitute another source of interference (Dyne et al. 1996; Dasgupta et al. 2000; Karppi et al. 2000; Marquet et al. 2010). Once more, individual laboratories need to validate the claim that these containers do not adsorb the XBs intented to analyze. The principles outlined by Bush et al. (2001) are a reasonable approach to such studies. It is important that the container size chosen for each specimen will allow it to be as close to full as possible in order to minimize concerns about oxidative losses due to air trapped at the top of the container, volatile XB volatilization, and ‘salting-out’ effects from preservatives that may be added to the tube (Moffat et al. 2004). Generally, 50-mL culture tubes represent the best choice for blood and urine specimens. Smaller tubes (e.g. 15, 20, and 30 mL) can be used for the collection of small amounts of blood, vitreous humor, and bile specimens. In Figure 2 an example of suitable sample containers is given. It is recommended to put sample containers inside another container for transporting. Finally, the correct collection of biological specimens for histological analysis is also important, since histology represents a very powerful tool in forensic analysis and for many times it has been used to corroborate other findings. In Forensic Toxicology, optical microscopy has been routinely used. After collection, specimens are usually sectioned into ~ 5 mm3 cubic pieces, fixed with 4% (v/v) buffered formaldehyde for 24 hours and subjected to routine procedures for paraffin histology (Pontes et al. 2008; Dinis-Oliveira et al. 2009c). Forensic toxicology request An example of a Forensic Toxicology request designed to accompany specimens to the laboratory is provided in Figure 3. Request forms should be filled in as complete as possible and submitted to the laboratory, together with the samples for analysis. The fields requested to fill up on this form are essential for an adequate analysis, and could save
time and expenses, by assisting the toxicologist to use the most useful methods of analysis and to interpret the results in the context of the case at a later stage. A copy of the postmortem report should be submitted as soon as it is available and a copy of any police sudden death report may also be useful. Particular concern should be paid to the safety. As a general rule, samples should always be treated as if they are infective and therefore must be handled with care, especially if originating from drug abusers. The major common risks are associated with tuberculosis, hepatitis B, and human immunodeficiency virus (HIV). Urine is the least likely to be infective. Staff in regular contact with potentially infective materials must be properly trained in the safe handling and disposal of biological samples and should be vaccinated against hepatitis B, poliomyelitis, tuberculosis, and tetanus, and possibly other diseases in specific countries. Sample handling should be performed with due attention to preventing droplets splashing into the eyes and minimizing aerosol formation (wearing eye protection and performing mixing and other procedures in a fume cupboard or microbiological safety cabinet, using either sealable centrifuge tubes or a centrifuge with sealable rotors). Screw-capped sample tubes are preferable to those with push-in stoppers as there is less risk of aerosol formation when opening the tube (Flanagan et al. 2007). Specimens preservation and storage Reliable qualitative and quantitative toxicological analysis is the basis of a competent toxicological judgment and consultation in Clinical and Forensic Toxicology. Unreliable results may lead to over-estimation or under-estimation of effects, to false interpretations, and to unwarranted conclusions (Peters et al. 2007). In the worst case, this might result in unjustified legal consequences or wrong treatment of the patient (Peters 2007). Depending on the conditions and duration of handling and storage, XB concentrations might have changed considerably since specimen acquisition or since the time of death. Specimens, especially if they cannot be analyzed immediately, should be stored at an appropriate temperature, with an adequate preservative in case of demonstrated need, and in a safe and suitable environment, only accessible to authorized staff, to ensure security and integrity. With this procedure, it is possible to minimize interferences with the analytical methods and errors on the interpretation of the results. In some circumstances, these considerations need also to be applied to the transport and continued storage of samples after analysis, sometimes for long periods, accordingly to local judicial rules. This is because any unpredictable matters may be disclosed by further investigation of the police. Until now, obligatory recommendations for specimen preservation and storage do not exist. Generally, once collected, specimens should be stored in tightly sealed and well filled (but do not overfilled) containers at 4°C for short-term and −20°C or preferably at −80°C for long-term (more than 1 week). Exceptions to this include hair and nail, which are stable at room temperature, and filter-paper adsorbed dried
Forensic toxicology 367 A
D
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B
C
E
Figure 2. Sample and mailing containers. (a) Tube of 10 mL capacity containing a fluoride salt (especially important for cocaine, carbon monoxide, ethanol, γ–hydroxybutyric acid, and cyanide) and ethylenediaminetetraacetic acid (EDTA, only in ante-mortem samples) for peripheral blood and the mailing container. (b) Tube of 30 mL capacity for cardiac blood, urine, and organs specimens, and the transport container. (c) Syringe of 2 mL 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 blood samples for specified analyzes if refrigerated transport and storage is not feasible (Patchen et al. 1983; Croes et al. 1994; Howe and Handelsman 1997). Sealing is particularly important in the interpretation of forensic results, especially when volatile XBs are to be analyzed. Whole blood should not be frozen, if plasma or serum is to be analyzed. For instance, blood collected for clinical purposes is usually centrifuged to separate serum or plasma for testing on clinical analysers. Once separated, the serum or plasma vials may not be sealed
again after analysis. That will allow ethanol and other volatile solvents to evaporate, and therefore should be taken into account if the sample is analyzed subsequently for forensic purposes, sometimes several days later. Post-mortem specimens, more than any other specimen, are likely to show some form of altered state because of either an extended post-mortem period before collection or inadequate or prolonged storage before analysis. Decomposition and eventual liquefaction of tissues occur during post-mortem periods and interfere with analytical results (Drummer
368 R. J. Dinis-Oliveira et al.
TOXICOLOGICAL REQUEST
Forensic laboratory logotype
Using capital letter, fill the request in a complete and accurate way..
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:________________
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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 SUSPECTED ETIOLOGY Driving accident Driver
No
Telephone:____________
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
Forensic toxicology 369 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 Loss of weight
Shivering
Vomiting
Thirst
Convulsions
Miosis
Blindness Mydriasis
Constipation Delirium
Cyanosis Coma
Jaundice
Sweating
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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*
Quantity
Date*
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.
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370 R. J. Dinis-Oliveira et al. 2004). These phenomena are very much dependent on the interval between time of death and discovery of the body, the ambient temperature, and other environmental factors. One day in a tropical or very hot environment can induce significant putrefaction, while weeks at freezing temperatures often results in little observable changes (Drummer 2004). The extent of these changes also varies significantly between XBs. The available data proves that the majority of XBs are stable in blood, plasma, or serum samples under the conditions usually encountered in a Clinical or Forensic Toxicology laboratory. Instability usually occurs for XBs carrying ester moieties, such as cocaine, heroin, or methylphenidate, containing sulfur atoms, such as phenothiazines or olanzapine, or other easily oxidized or reduced structures such as nitrobenzodiazepines or dihydropyridine-type calcium channel blockers. An excellent review on this topic was recently presented by Peters (2007). Specimen preservatives are generally not required for specimens other than blood. Besides a preserved blood sample, an unpreserved specimen is also desirable (Skopp 2004). Fluoride preservation with a final concentration of 1–5% sodium (or potassium) fluoride by weight is usually recommended. Only when a small amount of blood is collected, the excess fluoride may affect headspace volatile assays by altering the vapor pressure of the analyte (Prouty and Anderson 1987). Fluoride is mainly added to inhibit microorganismmediated conversion of glucose to ethanol, microorganismmediated oxidation of ethanol (Holmgren et al. 2004; Skopp 2004; Flanagan and Connally 2005; Jones 2006; Kugelberg and Jones 2007; Bendroth et al. 2008), post-mortem conversion of cocaine to ecgonine methyl ester by pseudocholinesterases (Isenschmid et al. 1989; 1992), enzymatic loss of other esters such as 6-acetylmorphine, and γ–hydroxybutyric acid (GHB) production (Ferrara et al. 1993; 1995; Karch et al. 2001) after death and in stored samples. Therefore, when interpreting forensic toxicological results, a serum or plasma specimen collected in the emergency department might be expected to contain higher concentrations of unchanged XB than a post-mortem blood sample collected later. However, this is often not the case, because post-mortem blood is regularly collected in tubes containing fluoride, which avoids alterations of XB concentrations, whereas clinical samples are typically unpreserved. If high amounts of glucose and marked contamination by bacteria are present, non-negligible amounts of ethanol can be produced in collected specimens. To discriminate ethanol produced post-mortem from the ante-mortem one, n-propanol can be used as an indicator, because it is concomitantly produced by bacteria. In that case, the concentration of n-propanol is not lower than 5% of a post-mortem ethanol concentration. The most typical amine produced during putrefaction is β-phenylethylamine. Its structure is similar to that of amphetamines. The similarity of the amine sometimes gives false positive results during screening by immunoassays (Moriya and Hashimoto 1997b). In analysis of XBs from specimens collected from polytraumatic victims, followed by intensive medical treatments, special caution is needed. In such victims, non-negligible
amounts of β-phenylethylamine are sometimes produced by the action of bacterial translocation (Border et al. 1987). On the other hand, fluoride preservation must not be used when organophosphorous chemicals are involved. In sodium fluoride preserved blood samples, dichlorvos is degraded completely within 15 min (Moriya and Hashimoto 1999a; Moriya et al. 1999). Esters, subject to alkaline hydrolysis, are more stable in post-mortem blood than ante-mortem blood because the pH of blood falls after death. Acidification may also stabilize some labile conjugates such as N-glycosides or cocaine (Moriya and Hashimoto 1996a). Ascorbic acid may be used as an antioxidant. For example, losses in olanzapine during storage at 4°C may be reduced by addition of 0.25% ascorbic acid (Olesen and Linnet 1998). However, the presence of an antioxidant may have reverse effects. During storage, the reduction has been observed of the N-oxide metabolites of chlorpromazine in samples containing antioxidants, artificially increasing the concentration of the parent drug (Curry and Evans 1976). An alternative way to preserve post-mortem fluids for qualitative analysis is the use of filter paper (Skopp 2004). This technique dates back to the early 1960s, when Dr Guthrie used dried blood spot specimens to measure phenylalanine in newborns. Dried blood spot specimens represent a valuable and cost-effective means for many clinical applications. In Forensic Toxicology, this technique has only tentatively been used in XB analysis (Chace et al. 2001). Up to now, chloroquine, theophylline, and lead have been measured from human blood collected and dried on filter paper (Mei et al. 2001). Analysis of blood spots dried on filter paper were shown to minimize the breakdown of cocaine and to largely preserve the profile of the parent XB and corresponding products of hydrolysis at the time of sampling. Due to dehydration, both the chemical and enzymatic hydrolysis of cocaine, benzoylecgonine, and ecgonine methyl ester could be stopped, and the analytical profile was ensured for 17 days (Skopp 2004). The preservation of forensic urine XB specimens as dry stains has also been investigated. XBs such as amphetamine, benzoylecgonine, morphine, and phencyclidine were stable under the storage conditions investigated. An exception was 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in urine spots, which degraded to 0 after 12 weeks of storage at ambient temperature (DuBey and Caplan 1996). In clinical settings, sodium azide (NaN3) or sodium metabisulfite are used as antimicrobial agents for urine samples. However, production of γ–hydroxybutyric acid occurred even in such preserved samples stored at 4°C or at a higher temperature (Kerrigan 2002). Some benzodiazepines, particularly the nitrobenzodiazepines (nitrazepam, nimetazepam, flunitrazepam, and clonazepam), are subject to post-mortem changes (Robertson and Drummer 1998). Nitrobenzodiazepines are actively converted to their respective metabolites by anaerobic (enteric) bacteria in the post-mortem interval (Robertson and Drummer 1995; 1998). Consequently, flunitrazepam is rarely confirmed positive in blood specimens taken at autopsy. Post-
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Forensic toxicology 371 mortem specimens collected for nitrobenzodiazepines determination should be analyzed as soon as possible to minimize the loss of their respective 7-amino-metabolites. Noteworthy, the 7-amino-benzodiazepines are less stable than the parent drugs at −20°C and require −60°C for reasonable stability (Robertson and Drummer 1998). Other benzodiazepines are also subject to post-mortem changes, but these changes can be minimized if specimens are stored at −20°C or lower and analyzed promptly (El Mahjoub and Staub 2000). Data suggests that many other benzodiazepines such as diazepam and temazepam are labile and are degraded under putrefying conditions (Levine et al. 1983; Stevens 1984). This means that these drugs may not be detected at all in decomposed samples (Drummer 2004). When extensive putrefaction has occurred and exposure to benzodiazepines is suspected, it is recommended to use other tissues where retention is more likely, such as hair (Drummer 2004). The stability of other drugs of abuse has been investigated (Moody et al. 1992; Hippenstiel and Gerson 1994; Romberg and Past 1994; Giorgi and Meeker 1995; Levine et al. 1996; Golding Fraga et al. 1998). Heroin and cocaine are probably the most notable examples. Heroin and cocaine are not only rapidly converted into their respective hydrolytic products in vivo, but also undergo rapid bioconversion in situ after death (Drummer 2004). Moreover, unless special precautions are undertaken, hydrolysis may even occur in the collection vessel. Heroin is converted within minutes to morphine through the 6-acetylmorphine intermediate. In fact, heroin is more susceptible to decomposition by plasma cholinesterase than cocaine, the half-life (t1/2) of the reaction in living subjects being only a few minutes. Therefore, it is difficult to detect heroin from the blood of a cadaver who had received intravenous injection only several minutes before (Goldberger et al. 1994); but 6-acetylmorphine, the main metabolite of heroin, is relatively stable in blood and detectable post-mortem (Goldberger et al. 1994). 6-acetylmorphine is relatively labile in urine undergoing deacetylation to morphine at room temperature, according to the pH of the specimen. However, 6-acetylmorphine is stable in frozen urine (−20°C) for at least 12 months (less than a 2% loss) (Fuller and Anderson 1992). Morphine is relatively stable in specimens when stored frozen, but shows significant losses when stored at 4°C or higher for more than a few days, or in post-mortem specimens (Carroll et al. 2000; Skopp et al. 2001). Of particular interest is the instability of morphine glucuronide conjugates. De-conjugation of morphine metabolites to morphine has been observed in liver (Moriya and Hashimoto 1997a). This issue has been recently discussed in the Shipman murders (Pounder 2003). Of further interest is the variability in morphine and morphine glucuronide ratios from different blood collection sites (Skopp et al. 1996). These data suggest that morphine and morphine glucuronide concentrations in the early stages of putrefaction, or when prolonged storage has occurred, may change substantially from the time of death. Cocaine is subject to chemical hydrolysis in the ante-mortem blood at pH 7.4 to benzoylecgonine, and, to a minor extent, it is metabolized by plasma cholinesterase (pseudocholinesterase) to yield
ecgonine methyl ester (Moriya and Hashimoto 1996c; Logan et al. 1997; Warner and Norman 2000). The pseudocholinesterase activity in blood barely declines 3 weeks after its storage at room temperature (Coe 1993). Ecgonine methyl ester is very rapidly further decomposed to ecgonine by chemical hydrolysis and thus does not accumulate in blood of living subjects (Moriya 2006). In the case of post-mortem blood, as mentioned above, the pH value rapidly declines due to anaerobic glycolysis, resulting in the absence of chemical hydrolysis of cocaine into benzoylecgonine, but instead on the accumulation of ecgonine methyl ester through the action of the coexisting cholinesterase. Therefore, the cocaine concentration in blood at the point of death may be estimated by summing up the concentrations of cocaine and ecgonine methyl ester (Isenschmid et al. 1992). While cocaine may be stabilized to some extent (degradation still occurs) by the addition of fluoride (a cholinesterase inhibitor)—2–3 weeks in a refrigerator—after blood collection, the extent of breakdown between death and autopsy must be considered (Baselt 1983). Dugan et al. (1994) showed that cocaine concentrations in urine can change by as much as −37% over a 12-month period when stored at −20°C, although other drugs of abuse are reasonably stable. While some loss of cocaine occurs in frozen specimens, it is not associated with formation of ecgonine methyl ester (Levine et al. 1996). Moderate losses for benzoylecgonine and 11-nor9-carboxy-Δ9-tetrahydrocannabinol have also been reported in urine stored frozen (Romberg and Past 1994; Levine et al. 1996). The concentration of the acid metabolite of Δ9-tetrahydrocannabinol, 11-nor-9-carboxy-Δ9tetrahydrocannabinol, shows significant decreases not only when urine is stored at room temperature for several days but also after long-term frozen storage (Romberg and Past 1994; Golding Fraga et al. 1998; Moody et al. 1999). Chain of custody One major difference between Forensic and Clinical Toxicology is that forensic work has legal repercussions. This implies that all evidence associated with a specific case must be kept in a secure area at all times and its lifetime must be documented by using a chain of custody record (Karch 2008). This documentation is central to the demonstration that the evidence has remained intact, and not been adulterated, changed, mishandled, or misplaced in any way that would compromise its integrity. Properly maintained chain of custody documentation rules out any period of time in which a specimen may be left vulnerable to adulteration or tampering (Karch 2008). Evidence ties together people, places, actions, and things that have important impacts on circumstances surrounding events in which individuals are held legally accountable. The procedure also raises the issue of confidentiality and ethics, where illegal activities may be involved (Wolff et al. 1999b). In criminal actions, the importance of the evidence may truly involve a ‘life or death’ sentence, while in civil litigation large sums of money or property may be at stake. Failure to properly document the chain of custody may
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372 R. J. Dinis-Oliveira et al. compromise not only the integrity of the specimen, but also the credibility of the institution handling the specimen. Limited-access areas, at all times, with restricted access to only those individuals designated in the institution’s standard operating procedure, are mandatory. The chain of custody data should include the dates and identification of individuals performing the sample collection, transportation to the laboratory, and confirmation of receipt. Basically, the chain of custody should be able to answer: who handled the evidence, what evidence was handled, when and why the evidence was handled, and where the evidence was located at all times (Karch 2008). Whenever the specimens are left unattended, they should be secured in a locked container, refrigerator, or freezer. An example of a chain of custody report is given in Figure 4. In order to fulfill the chain of custody demands, samples and toxicological request should be put inside an opaque plastic bag sealed in such a way that any evidence of tampering (tamper-proof containers) would be evident.
General aspects of ante-mortem forensic toxicology When an intoxication incident occurs and the victim is sent to an health unit, medical doctors and co-medical staff should concentrate their efforts on the patient intensive care (Suzuki and Watanabe 2005), irrespective of any other aspects surrounding the case. Medical doctors and comedical staffs should communicate with the Local Poison Information Center, giving as much information about the patient as possible, namely clinical data (since databases have been developed for estimation of a causative XB according to clinical data), but also results of clinical tests, any previous medical history of poisoning, details of drugs or other XBs to which the patient may have had access, and,
Forensic laboratory logotype
in cases of accidental poisoning, XBs to which the patient may have been exposed (Moffat et al. 2004). The information exchange should be made between the doctor directly treating the patient and staff of the Local Poison Information Center or any expert toxicologist for the specific XB. Their conversation, aimed to clarify symptoms, often yields clues for the cause of toxicity and for the priority of the analysis to be performed. Close communication must continue if the initial tests prove negative, so that the search can be widened, or if the clinician requires advice on the interpretation of positive results (Moffat et al. 2004). When the patient’s condition is severe and the diagnosis is not clear, toxicological tests may be crucial. Analytical toxicological results must be rapidly supplied (usually within 1–2 hours of the patient’s arrival) without losing accuracy. Preliminary screening tests may be required to narrow causative XB candidates before accurate analysis by instruments of high performance. The identification of a causative XB is one of the most important tasks in emergency medicine. Ideally, the XB can be both identified and quantified within this time frame. When this is not possible, a qualitative result still has considerable value if the symptoms are consistent with the identified XB and should be given to the clinician without delay (Moffat et al. 2004). However, they must be of sufficient quality to allow an appropriate clinical decision to be made. It is important to discuss the scope and limitations of the tests performed with the clinician and to maintain high standards of laboratory practice, especially when performing tests on an emergency basis. It may be better to offer no result rather than misleading data based on unreliable tests (Flanagan et al. 2007). Though clinicians often treat poisoned patients on the basis of clinical history rather than wait for toxicological results, they may change their approach once they have them. In addition, the analysis should not end after the first positive finding because
CHAIN OF CUSTODY REPORT
Using capital letter, fill the chain of custody report in a complete and accurate way.
VICTIM IDENTIFICATION Name:_______________________________________________________ Birth date:______________________ Age:__________ Male
Female FROM
TO
Name:_______________________________________ Date/Time of collection
transport
:____________
Nº of the specimen handled: Why it was handled?___________________________ Name:_______________________________________ Date/Time of collection
transport
:____________
Nº of the specimen handled: Why it was handled?___________________________
Confirmation of receipt:_________________________ Date /Time:___________________________________ Nº of the specimen handled: Why it was handled?___________________________ Confirmation of receipt:_________________________ Date /Time:___________________________________ Nº of the specimen handled: Why it was handled?___________________________
Figure 4. Example of a chain of custody report. Rows ‘from–to’ should be repeted if necessary.
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Forensic toxicology 373 additional, hitherto unsuspected XBs may be present. Of course, a ‘positive’ result on a ‘poison screening’ does not by itself confirm poisoning, because such a result may arise from incidental or occupational exposure to the XB in question or the use of drugs in treatment (Flanagan et al. 2007). Frequently routine clinical chemical tests will be performed at one site, while more complex toxicological analysis will be performed by a different department, possibly at a different location. Screening tests could be performed at bedside or in the Clinical Laboratory, inside Health Care Unit, whereas complex qualitative and quantitative analysis are usually performed at laboratories with more accurate analytical instruments and specialized analysts, such as local academic institutions or institutes of legal medicine. However, the range of analyses that can be offered by specialized laboratories to meet emergency demands usually encompasses only a few hundred XBs. Fortunately, in the vast majority of cases, the diagnosis can be made on circumstantial and clinical evidence (Moffat et al. 2004). The analytical toxicological requests to institutions besides forensic ones is only possible when a poisoned patient is alive and no criminality signs exist. Accidental self-poisoning and attempted suicide cases are generally under the responsibility of the clinical toxicologist. Only a small proportion of these cases should be referred to the forensic toxicologist if there are suspicions of criminality. Criminality cases include: a. Iatrogenic poisoning, in which a patient or relative sues a health authority and its staff for neglection, because the patient dies and a coroner’s inquest is ordered (Moffat et al. 2004); b. Victims of so-called date-rape, who were administered XBs such as flunitrazepam or γ–hydroxybutyric acid to induce confusion and amnesia and facilitate sexual abuse, and elderly abuse (Bechtel and Holstege 2007; Kintz et al. 2008); c. Non-accidental poisoning in children. Mothers are the most frequent perpetrators of child poisoning and do so to attract sympathy and attention as a consequence of the child’s illness (Münchausen’s syndrome by proxy) (Meadow 1977; 1998; Bader and Kerzner 1999; Bappal et al. 2001; Aranibar and Cerda 2005; Carter et al. 2006; Holstege and Dobmeier 2006). When these situations arise, the hospital toxicologist is obliged to take special precautions to conserve all residual samples and documentation that may feature subsequently as part of a forensic investigation; d. Driving under the influence of alcohol or psychotropic substances; e. Issues relating to persons subject to disqualification by their usual consumption of alcoholic beverages and psychotropic substances; and f. Doping in sport and workplace drug testing (Christophersen and Morland 1994; Botre 2003; Maravelias et al. 2005; Centini et al. 2007).
Since the preliminary analysis may have been already carried out, close co-operation between the forensic and hospital laboratories is desirable (Moffat et al. 2004). Frequently, victims of intoxication are admitted to hospital, even if they survive only briefly, such as occurrences in traffic accidents or overdoses. Even if death arises fairly soon after admission to hospital, it is common that blood, urine, and gastric content are collected as part of the medical evaluation and treatment. Analysis of ante-mortem hospital admission specimens is invaluable, for several reasons (Moffat et al. 2004), namely: a. Gives a good idea of the circulating blood concentration of the putative XB at the time of admission to the hospital, which by definition is unaffected by post-mortem redistribution; b. May provide the only reliable indicator of dosage; and c. May provide the unique opportunity to perform meaningful toxicology, if the person survives long enough for a XB to be eliminated from the body prior to death, or to be diminished to a concentration of limited or no forensic value. Even if blood or plasma collected on admission is not available, clearly timed specimens drawn several hours later may still be useful if allowance is made for clearance and for the presence of drugs administered as part of treatment.
General aspects of post-mortem forensic toxicology Any violent, unnatural, sudden, or unexpected death should be investigated to establish the cause and manner of death. In other words, any death that cannot be explained by a medically recognized disease should be referred to the pathologist for investigation, and Forensic Toxicology represents a strong ally. The aim of Forensic Toxicology is to help establish the role that XBs played in a death, or in events immediately before death. It is the greater difficulty of interpreting postmortem results that most differentiates post-mortem Forensic Toxicology from ante-mortem Forensic Toxicology. In general, a case falls under the jurisdiction of a legal medicine institute when the death (Stripp 2006): a. Results from violent, criminal, suicidal, or accidental means, including any death from criminal neglect or due to suspicious or unexplained activity; b. Occurs in an apparently healthy person with no explained causes (indeterminate death); c. Occurs during certain medical procedures; d. Raises doubts if the victim is a fetal or a stillbirth case; e. Occurs with no attending physician present; f. Occurs when the victim is incarcerated or confined; or g. May have been caused by XBs poisoning. Specimens are rarely ideal in post-mortem cases and without specialist knowledge any results should be considered with
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374 R. J. Dinis-Oliveira et al. caution when attempting to interpret their significance. If the decedent was hospitalized prior to death, ante-mortem specimens, if available, should be submitted for analysis. In addition, existing ante- or peri-mortem (at or near the time of death) specimens do not negate the need to collect postmortem specimens. The development of gas chromatography (GC) and highperformance liquid chromatography (HPLC) during the early 1970s had a major influence on the development and growth of pharmacokinetics and therapeutic drug monitoring (Karch 2008). As a result, the kinetics of XBs in clinical patients was easier to understand and predict. It was logical that toxicologists started to use the pharmacokinetic data obtained from living patients to interpret post-mortem blood concentrations, for example, to predict whether a given blood XB concentration was ‘in the therapeutic range’, whether the blood level was ‘fatal’, or even to predict the amount ingested prior to death (Karch 2008). Experience has since shown that post-mortem XB concentrations must be interpreted from a very different perspective relative to those in living patients. Indeed, most XBs measurements in samples from living patients are made on plasma or serum, and whole blood is used uncommonly (Ferner 2008). In addition, many processes (often refereed as artifacts) occur after death that can change XB concentrations, sometimes to a very large extent, which must be known in order to interpret results (Karch 2008). The presence of putrefactive changes of specimens limits the direct applicability of clinically validated assays in a post-mortem setting. In addition, several alternative specimens can be collected in a post-mortem setting. The period of enthusiasm in the late 1970s and 1980s has given way to the realization that there are many unique aspects of post-mortem toxicology that must be considered when interpreting analytical results (Karch 2008). It is no longer acceptable (indeed it is impossible) to interpret post-mortem toxicology results from tables of so-called therapeutic, toxic, and fatal ranges, without taking into consideration the medical and case history, presumed dose, autopsy findings, information from the scene, the immediate circumstances of the death, the various processes that can affect XBs concentrations both before and after death, and the exclusion of other potential causes. For instance, a Swiss study has suggested that medical history plays an important role in interpreting post-mortem data in ~ 70% of cases (Harding-Pink and Fryc 1991). It is probably fair to say that many toxicologists and pathologists are less confident about interpreting post-mortem XB concentrations today—comparatively to 10–20 years ago.
Collection of biological specimens for toxicological analysis In analytical toxicology, clinical chemistry and related fields, the words ‘sample’ and ‘specimen’ are used to denote a portion of a body fluid, tissue, incubation medium, etc., collected under defined conditions (Flanagan et al. 2007). Up to date, a harmonized protocol for sampling in suspected poisoning
or XB-related intoxications has not yet been established, but it is a broad consensus that collection should be performed as early as possible after intoxication. It is also obvious that sampling procedure, selection of specimens, or quantity for toxicological analysis has to be case-dependent (Plebani and Carraro 1997), largely considering the case history, requests, legal aspects, and availability. Any specimen that was in contact with a XB is a potential candidate for toxicological investigations, at least for qualitative analysis. Although toxicology analysis can theoretically be performed on almost any specimen, it is usually limited to those for which there is an appropriate literature available to help in the interpretation of the results or for which there is a validated method (Moffat et al. 2004). Blood, urine, and gastric content, namely gastric lavage fluid and vomitus, are normally used as specimens for XB analysis in living subjects. In post-mortem toxicology investigations, available specimens can be numerous and variable (Skopp 2004). They may range from relatively pure solutions of a XB to a putrefying specimen. Proper collection and preservation of post-mortem specimens is critical, since there is usually no opportunity to go back for re-collection of specimens, as occurs in cremation. Generally, the specimens routinely collected at autopsy include fluids such as peripheral and cardiac blood, urine, bile, cerebrospinal fluid, vitreous humor, and gastric content and organs, particularly liver (Forrest 1993). Kidney, brain, lung, spleen, and skeletal muscle are also occasionally collected for post-mortem studies. Blood from peripheral sites should be obtained from femoral vein and prior opening the thorax and abdominal cavities (Skopp 2004). Samples that may also be collected prior to organ dissection are urine, bile, cerebrospinal fluid, or fluid from putrefactive blisters, hair, nails, and swabs from the orifices of the body or from the skin (Skopp 2004). Organs are normally collected after evisceration. They provide one of the best and most useful specimens to assist in the interpretation of blood findings. XB detection in tissue specimens should be considered whenever highly lipophilic or preferably bound to tissue XBs are suspected to be involved in the intoxication. Organs may also be useful in cases with extended post-mortem time periods, namely in decomposed bodies, and whenever body fluids are not available or difficult to obtain. A large amount of data for XB findings in tissue exists, primarily for liver and kidney, and, to a lesser degree, brain and lung (Baselt 2004). The analysis of tissue is normally performed by weight, and therefore organ weights must be recorded. Usually, 1–4 g of tissue is homogenized 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 quantitative measurements are needed. Urine is commonly used in qualitative analysis since relatively large volumes are usually available (especially ante-mortem). The concentrations of many XBs, and their metabolites, tend to be higher than in blood, thereby facilitating detection. Gastric content can also be useful specimens for identification of a XB when the time after ingestion is short, since there are higher probabilities to
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Forensic toxicology 375 contain large amounts of an unchanged form of an ingested XB. For the other specimens available, qualitative importance should also be considered, instead of quantitative analysis (Drummer 2004). If an intoxication case is identified, additional questions may arise such as the route of administration and an acute or long-term exposure to a XB. In several circumstances and since there is a marked development of analytical technologies, additional and alternative specimens such as hair, nails, sweat, or skin samples could be collected to complete the toxicological investigation, because many XB are excreted and accumulated into these compartments. If subcutaneous or intravenous XB application is suspected as the cause of death, a sample of the particular skin region should be excised together with a random specimen preferably taken from a similar (but different) site to act as a control (Skopp 2004). A skin specimen or a cube of muscle taken from a suspected injection site may support evidence of that route of XB administration (Baselt 2004). Decomposed, skeletonized, or embalmed cases represent unique challenges for the toxicologist, due to the limited availability of specimens. In these cases, muscular tissue, hair, and bone are normally collected (Forrest 1993). In some cases, analyses of XBs in fly larvae, in decomposing bodies, provides an insight to the presence of XBs in the corpse. Soil samples collected at the site of a skeleton or decomposed body, and even cremation ash are also possible specimens to be considered (Vass et al. 2008; Lombardi 2009). Regarding the amount to be collect, there are no clear directives for sample sizes, but that should permit sufficient and sustainable conclusions to be made. Submission of very small samples may result in reduced sensitivity and scope of the analyses undertaken, but nevertheless such samples should always be forwarded to the laboratory. General requirements for sampling and the relative merits of each specimen are discussed in the following sections, and a resume is provided in Table 1. Whole blood, plasma, and serum Ante-mortem blood specimens are generally collected from the vein (usually the median cubital vein) using either a hypodermic needle and syringe (1–50 mL) or a commercial vacuum-sampling system (Flanagan et al. 2007). A tourniquet can be used to distend the vein prior to venepuncture, but should be released immediately prior to sampling. Disinfectant swabs that contain alcohols or iodine used to clean the skin prior to venepuncture can contaminate blood samples and should not be used, especially when performing ethanol analysis. In view of the requirement to prevent stress on patients, 5–10 mL of blood is enough. If the situation permits, multiple samplings at different intervals are desirable. For repeated sampling, a small cannula may be inserted into a vein in the arm or hand, which allows venous access via a rubber septum. If blood has been collected into a syringe, it is essential that the syringe needle is removed and the blood allowed to flow gently into the collection tube in order to prevent hemolysis. This should be followed by gentle mixing
to ensure contact with the anticoagulant if one is being used. Even mild hemolysis will invalidate a serum iron or potassium assay, and plasma or serum assays for other analytes concentrated in red cells such as chlortalidone (Fleuren and Van Rossum 1978; Guder 1986; Sonntag 1986; Delanghe 2000). EDTA, citrate, or heparin are currently the used anticoagulants. Sodium citrate is normally used for clotting studies. Since tubes contain 0.5 or 1 mL of the anticoagulant in aqueous solution, quantitative analysis is not suitable (Flanagan et al. 2007). Furthermore, dilution of the sample may reduce the degree of plasma protein binding and consequently the plasma:red cell distribution of the analyte. It should also be ensured that lithium heparin anticoagulant is not used if plasma lithium is to be measured (Flanagan et al. 2007). If plasma or serum is required, these specimens should be separated from blood cells as soon as possible. Plasma is obtained by centrifuging the tubes containing anticoagulated whole blood at 2000–3000 g for 10 min and at 2–8°C if necessary. If whole blood is allowed to stand (15 min, room temperature) in a tube without anticoagulant, a clot forms that will retract sufficiently to allow serum to be collected (Flanagan et al. 2007). More plasma than serum can be separated from whole blood. To collect erythrocytes, heparinized blood should be centrifuged (2000 g, 10 min), the plasma, buffy coat, and top 10% of erythrocytes (mainly reticulocytes) removed, and the remaining erythrocytes carefully washed with isotonic, buffered saline to remove trapped plasma. The cells may be used directly or frozen, either to cause hemolysis, or for storage. Platelets are usually isolated by the slow centrifugation (e.g. 300 g, 15 min) of anticoagulated whole blood to yield platelet-rich plasma, which is recentrifuged (2000 g, 10 min) to harvest the platelets. Other white blood cells are most commonly obtained by centrifugation through media of appropriate density (according to the manufacturer’s instructions) or isolated by solid-phase antibody techniques (Flanagan et al. 2007). Arterial blood is normally collected by an experienced medical practitioner (it is a relatively dangerous procedure) for the measurement of blood gases and is not usually used for toxicological analysis. Capillary blood, which closely approximates to arterial blood, can be obtained by pricking the heel, finger, or ear lobe; this procedure is most often performed on small children. Ante-mortem, most quantitative assays are carried out on the plasma and/or serum (Chamberlain 1995), but anticoagulated whole blood is essential if the XB is mainly associated with the red cells (e.g. carbon monoxide, cyanide, lead, mercury). If necessary, whole blood can be stored at −20°C or below, but freezing will lyse most cell types. Some XBs, such as many benzodiazepines, are extensively metabolized prior to excretion and then plasma is the specimen of choice for detecting the parent XB. Leaving plasma or serum in contact with red cells can cause changes due to enzymatic activity or redistribution of an analyte between cells and plasma. Serum from coagulated blood can be used for the majority of the cases since the levels are almost always the same as
376 R. J. Dinis-Oliveira et al. 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 -30 mL 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.
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BLOOD CLOTS FROM SUBDURAL, SUBARACHNOID, AND/OR EPIDURAL SPACES -30 mL for plastic universal container with screw cap; -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 -30 mL for plastic universal container with screw cap; -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 -10 mL for plastic universal container with screw cap; -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 10 mL 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 -10 mL for plastic universal container with screw cap; -Always collected for complete toxicology analysis; -Adult human body contains ~ 5–6 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
Forensic toxicology 377 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.
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URINE -30 mL or all available for 30 mL plastic universal container with screw cap (no preservative); -Always collected; -Submit any quantity, even if lower then 1 mL, 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 -30 mL of the total homogenized for 30 mL 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 10 mL 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 elements 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 -30 g for a 30 mL 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 10 mL 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 on next page
378 R. J. Dinis-Oliveira et al. 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.
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SPLEEN -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 -30 g for a 30 mL plastic universal container with screw cap (no preservative); -Collected for metals or ethylene glycol analysis; -Remove capsule; -Could be important in absence of urine. HEART -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 50 mg); -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 identical 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, depending 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 on next page
Forensic toxicology 379 Table 1. Continued. -The risk of external contamination leads to report false positive results; -Not a suitable specimen for detecting recent XB use. BONE -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 30 mL 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;
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-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 -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 concentrations 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 -30 g for a 30 mL plastic universal container with screw cap (no preservative); -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 -1-2 mL 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 (2 g minimum) for a 10 mL plastic universal container; Table 1. continued on next page
380 R. J. Dinis-Oliveira et al. 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 72 h.
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OTHER SAMPLES -Faeces; -Nasal swabs (fluid collected onto cotton swabs from inside the nose), peritoneal (fluid that accumulates in the peritoneum) and bronchoalveolar lavage (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 it occurs in paraquat serum concentrations that are ~ 3-fold lower than those in plasma obtained from the same blood sample (Dinis-Oliveira et al. 2008). For many analyses, serum is preferred to plasma because it produces less precipitate (of fibrin) on freezing and thawing and has the advantage that there is no potential interfering additives. Its composition is generally the same as plasma, except that fibrinogen and factors associated with the clotting process are absent. Post-mortem, whole blood is the specimen of choice for detecting, quantifying, and interpreting XB concentrations since it is relatively easy to collect and most of the meaningful data derived from the literature was determined in serum, plasma and sometimes in whole blood (Leikin and Watson 2003; Baselt 2008). Therefore, blood should always be collected. Nevertheless, it is important to take into account that the specimen collected as ‘blood’ at autopsy is not the same collected in an ante-mortem venipuncture, and therefore clinically-based kinetic principles (mainly derived from serum and/or plasma) may not be directly applicable to post-mortem cases (Karch 2008). Indeed, blood obtained post-mortem is a very variable sample. Normally it is considered to: a. Be relatively fluid but more viscous (may be clotted or completely fluid or partly clotted and partly fluid); b. Have typically numerous small clots (anticoagulants are not recommended for post-mortem blood samples because these additives may affect XB concentration. For instance, blood concentration of morphine in EDTA tubes was 4.8% higher than in heparin tubes (Westerling et al. 1996)); c. Possess sedimented cells; d. Be contaminated with tissue fluid before collection; e. Be potentially putrefied before collection; f. Have a pH up to 5.5 (a sharp decrease in pH occurs immediately after death, which again slowly increases during the post-mortem interval due to the breakdown of protein);
g. Range from 60–90% in terms of water content; h. Be potentially dehydrated from exposure to heat during a fire; or i. Have a high degree of hemolysis, and for this reason whole blood is normally analyzed directly. Ensuring that the body is stored at 2–4°C prior to the autopsy, and that it is processed as soon as possible after death, will minimize the risk of changes in blood analyte concentrations occurring before sampling. The general rules that the site or source of specimen collection should always be clearly stated on the specimen container and specimens taken from different sites should never be combined but always submitted separately, acquires more post-mortem importance, since post-mortem redistribution can cause the concentrations of many XB to vary markedly from site to site (Jones and Pounder 1987; Pounder and Jones 1990; Prouty and Anderson 1990; Pounder et al. 1996a). Blood, simply labeled as such, could come from almost anywhere— for instance as pooled blood at the scene. Even XB concentrations in blood drawn from the same site, but simply placed into different collection vials, can also sometimes differ by several fold (Karch 2008). Cardiac blood is usually more abundant than peripheral blood, and XB levels in heart blood are generally higher than in femoral venous blood. Therefore, post-mortem screening toxicological tests may preferentially be performed on a heart blood sample in opposition to urine, especially for XBs that are extensively metabolized, reserving the peripheral blood specimens for cases where additional context is needed for interpretation. To obtain a proper cardiac specimen, whenever possible, the pericardial sack must be opened, the pericardium removed, the heart dried, and the blood specimen removed using a syringe, preferably from the right chamber (Figure 5) (Karch 2008). It is essential to label the site from which it was taken (right or left heart chamber) or to provide the information on mixed heart blood. Most toxicologists and pathologists are well familiar with the widely discouraged practice of drawing blood by a ‘blind stick’ through the chest
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Forensic toxicology 381 A
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Figure 5. To obtain cardiac blood, pericardial sack is opened and removed (a–c) and a specimen is aspirated using a syringe, from the right chambe (d).
wall (Karch 2008). Although such blood may be labeled as ‘heart blood’, it may contain pericardial fluid and/or pleural fluid. Since it could come from the pleural cavity, it might therefore be contaminated by gastric content, particularly if the death was traumatic or decomposition severe (Logan and Lindholm 1996). Even blood drawn from the ‘heart’, after opening the body cavity at autopsy, may contain blood from a number of sources, such as: a. Blood from one or more of the cardiac chambers; and b. Blood that was drained from the pulmonary vein and artery (and hence the lungs), from the inferior vena cava (and hence from the liver), and from the aorta and subclavian veins. Accordingly, the so-called ‘heart blood’ is potentially one of the most non-homogeneous specimens in the body (Karch 2008). Post-mortem blood for quantitative analyses is preferably obtained from the femoral vein prior to autopsy to avoid contamination with stomach contents and small pieces of tissue(s) (Figure 6). Since many XBs are very potent, that is, the blood concentrations associated to severe or fatal intoxications are very low (typically mg/L or even μg/L), even trace contamination of a peripheral blood sample can confound the most careful analytical work. In such instances, toxicological analysis can often do little more than provide evidence of exposure to a particular XB (Flanagan and Connally 2005). Leg veins are preferred to veins of the head and neck due
to the anatomical presence of a larger number of valves that resist blood movement from the intestines (Harper and Couy 1988). Therefore, this site is usually less, but not completely, affected by post-mortem changes in XB concentration, since redistribution from the bladder to femoral venous blood concentrations was observed (Moriya and Hashimoto 2001). Duplicate blood samples from distinct peripheral sites, e.g. the right and left femoral vein, may be taken to assure that XB concentration had remained fairly constant post-mortem. The peripheral blood specimen should be taken using a clean or new 10–20-mL hypodermic syringe. The leg should not be massaged in order to increase specimen volume. While it is certainly a good practice to obtain, wherever achievable, peripheral blood, to avoid as much as possible the effects of post-mortem redistribution or diffusion from the major organs, interpreters should be cautioned that there is no such thing as ‘pure femoral blood’ (Flanagan and Connally 2005). It is simply blood drawn from the site of the femoral vein. Surely, if the proximal part of the femoral vein is clamped prior to sampling, it is likely that much of the blood will be ‘peripheral’ and therefore relatively uncontaminated by blood from the major organs. Therefore, to obtain femoral blood, some forensic toxicologists advocate clamping the femoral vessels (Figure 6) (Yarema and Becker 2005). While in many of the published studies on post-mortem redistribution the vessels have been carefully clamped prior to taking blood samples, this is rarely done during routine medicolegal autopsies, since this procedure results in added time to the autopsy examination as well as added incisions. Typically,
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382 R. J. Dinis-Oliveira et al. femoral blood is drawn by a ‘stick’ to the unclamped femoral vein in the groin area. In order to tie off the femoral vein, medical examiners must perform a ‘cut-down’ incision over the proximal thigh to expose the vessel fully and the leg could be slightly elevated to obtain more blood (Figure 6). If the volume drawn is relatively small (e.g. 2–5 mL), it is unlikely that much blood will be drawn from the central body cavity, since it is also difficult to collect more than 5–10 mL from a clamped femoral vein (Karch 2008). Without ligation, at least some blood will have been drawn down from the inferior vena cava, and hence from the liver, and from the larger iliac vein. Notwithstanding these possibilities, Hargrove and McCutcheon (2008) showed good correlation between blind stick femoral and clamped femoral samples for eight drugs from four different drug classes. These authors concluded that a blind stick femoral blood sample does not have significant redistribution from central sites and is of equivalent quality to a clamped femoral sample. An alternative sampling technique is to cut the iliac vein at the side of the pelvis during autopsy, and to only sample blood that is massaged out from the femoral vein directly into a test tube. Even if such a procedure ensures that the collected blood is from the femoral vein, some post-mortem changes may just as well have happened in this blood too, e.g. diffusion from vessel walls and skeletal muscle. Since blood concentrations of some XBs may change markedly post-mortem, some authors advocate to analyze blood obtained from more than one site, plus tissue or other
specimens where this may be useful (Jones and Pounder 1987; Pounder and Jones 1990; Prouty and Anderson 1990; Pounder et al. 1996a). In some cases, e.g. in severe trauma, a peripheral specimen may be collected from the arm, specifically from the subclavian vein. Baselt (2008) and Leikin and Watson (2003) provided most comprehensive data on a comparison of XB concentrations in post-mortem cardiac and femoral venous blood. Many XBs displayed a wide range of ratios of XB concentrations in cardiac vs peripheral blood. Important factors affecting the cardiac vs peripheral blood ratio are the type of XB, its volume of distribution, concentration range, protein binding, pKa-value, and the post-mortem interval between death and autopsy. Generally, basic XB with a large volume of distribution showed the greatest range in cardiac vs peripheral blood ratios as well as the largest ratios. Blood clots and ‘blood’ from thoracic and abdominal cavities It is often common that a victim survives for several hours after a fall or blunt trauma to the head, with circulation remaining intact until the time of death (Kugelberg and Jones 2007). Owing to the reduced circulation in the damaged region of the brain, XBs in the blood clots (e.g. subdural, subarachnoid, and/or epidural) are not metabolized to the same extent as in blood circulating through the liver (Kugelberg and Jones 2007). Accordingly, the blood clot
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Figure 6. To obtain femoral blood, medical examiners must perform a ‘cut-down’ incision over the proximal thigh (a–c) 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.
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Forensic toxicology 383 will contain a higher concentration of XBs compared with a specimen of peripheral venous blood obtained at autopsy. Therefore, the sampling and analysis of intracranial blood clots might furnish useful complementary information about the person’s blood XB concentration several hours before death, such as when the trauma/injury occurred. As a result, blood clots are considered to be potential ‘time capsules’ (Karch 2008). Nevertheless, it must be pondered that an injury often results in destruction of the skin surface and surrounding tissue, which means that bacteria and infection can enter the wound, increasing the potential for microbial synthesis of ethanol in the blood clot after death. The usefulness of analysis in intracranial blood clots and comparisons with concentrations in peripheral or heart blood at autopsy has been reported before, especially for ethanol (Moriya and Hashimoto 1996b; 1998b; Riggs et al. 1998; Takahashi et al. 1999; 2001; Kugelberg and Jones 2007; Boonyoung et al. 2008). Blood clots may also be useful for documenting pre-existing XB use prior to hospital therapy. Following severe injury or trauma, thoracic (pleural) and abdominal cavities ‘blood’ should be collected for analysis only if blood or uncontaminated blood clots cannot be obtained from any other area. The composition of these ‘blood’ specimens markedly differs from whole blood due to the strong possibility of contamination by microorganisms or with gastric and/or intestinal contents resulting from severe trauma. For example, it is not uncommon that severe motor vehicle accidents result in rupture of the stomach and diaphragm. If an autopsy is performed, the origin and nature of the fluid so drawn should be obvious, and hopefully noted (Karch 2008). However, if an autopsy is not performed and ‘blood’ is sampled through the chest wall in an attempt to obtain cardiac blood, the coroner or medical examiner should be conscious that the sample is almost certainly contaminated with gastric content. Trauma causing extended blood loss may also affect blood XB levels due to the physiological reactions, namely the increased heart rate and peripheral vasoconstriction, and plasma volume refill therapies. Hence, blood XB levels may increase or drop, depending on their concentrations in the restoration fluid. Experimentally, codeine and morphine blood levels were found to increase significantly after controlled exsanguination in rats (Kugelberg et al. 2003; Jones et al. 2008) and a similar study showed that the analgesic effect of morphine incresed when given to rats with hemorrhagic shock (De Paepe et al. 1998). Although further studies are needed to determine the influence of such conditions on ante-mortem redistribution for several XBs with different pharmacokinetic properties, the phenomenon should be considered in trauma cases with longer duration of blood loss. Nevertheless, qualitative documentation of the presence of given analytes is of importance and value in death investigations with respect to compliance and exposure issues (Karch 2008). Therefore, these ‘blood-like’ samples only provide a qualitative documentation of the presence of a XB.
Urine Urine represents one of the major routes to eliminate XBs from the body. The accumulation of parent XBs and their metabolites in urine usually results in high concentrations, facilitating their detection. It is mostly used as a screening specimen (thought it is not always available), e.g. in death related to drugs of abuse and prescribed medication as well as in apparent accidental death where impairment is suspected (Drummer and Gerostamoulos 2002). The urine matrix is generally devoid of circulating serum proteins, lipids and other related large-molecular-weight compounds due to the glomerular filtration process. These characteristics greatly simplify the preparation of the specimen for toxicological analysis, enabling the investigation either directly by immunoassays or non-instrumental spot tests as well as after extraction with an appropriate solvent (Skopp 2004). Detection times for XBs in urine can vary from 24 h to as long as a month, depending on the compound (Karch 2008). In cases where death is suspected to have occurred rapidly due to XB exposure, as might be suggested by the presence of a needle in the decedent’s arm at the time of death, negative urine findings are possible, and consistent if blood concentrations of the analyte are very high. Thus, except for these acute XB deaths, where survival time is less than 1 h (may not yet have been excreted into the urine), urine provides an ideal matrix for the detection for the widest variety of XBs. It is almost universally accepted that, with few exceptions, there is very little correlation between urine and blood XB concentrations, and even less correlation between urine XB concentrations and pharmacological or toxicological effects. Therefore, quantitative measurements in urine are generally of little use in toxicology (Suzuki and Watanabe 2005). To interpret the context of exposure, blood should be tested for the analytes found in the urine (Karch 2008). Indeed, many factors affect urine concentration, such as fluid intake, rate of metabolism, glomerular clearance, urine pH (weakly basic compounds such as amphetamine or methadone are more efficiently excreted in acidic urine, whereas weakly acidic compounds, such as barbiturates, are more efficiently excreted in basic urine (Wolff et al. 1999a)) and the times of voiding relative to the dose. Therefore, any attempt to predict or even estimate a blood concentration from a urine concentration is pure imprudence. The wide variation in its composition can be corrected by the creatinine value of the particular sample (Skopp 2004). A volume of 30 mL or all available urine (no preservative) is sufficient for most purposes and samplings obtained at time intervals are preferable. When urine is obtained by catheterization from a patient, it should be considered the possibility of being contaminated with a local anesthetic that was applied to the catheter as a gel formulation. During autopsy, urine specimens should be taken by insertion of a clean/ new hypodermic needle into the bladder (Figure 7) (Karch 2008). For victims not subjected to internal examination, the needle may be inserted directly through the lower abdominal wall, just above the pubic symphysis (Figure 7) (DiMaio and DiMaio 2001). In cases where the bladder appears to
384 R. J. Dinis-Oliveira et al.
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Figure 7. To obtain urine, for victims not subjected to internal examination, 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).
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be empty, it is important to aspirate as much urine as possible 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 container 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, namely those that undergo biliary excretion by being substrates of P-glycoprotein efflux transporter and are often subject of enterohepatic circulation (Elferink et al. 1995; Fardel et al. 2001; Dinis-Oliveira et al. 2006b; 2008). It is a useful fluid for qualitative analysis and can be used in screening when urine is not available. Biliary XBs concentrations may also be influenced by post-mortem diffusion from the liver and the stomach (Karch 2008). Bile is aspirated from the gallbladder using a clean/new hypodermic syringe (Figure 8). Due to its complexity (it contains high concentrations of bile acids and other endogenous substances), methods developed for other fluids with well-established consistency may not be immediately adaptable to bile (Agarwal and Lemos 1996). Because XBs concentrations are often higher in bile than
Figure 8. Bile is collected prior to liver. Gallbladder is exposed (a), should be tied to reduce contamination, and bile is aspirated using a clean/new hypodermic syringe (b, c).
corresponding blood specimens (Vanbinst et al. 2002), the use of a smaller sample volume and dilution into a buffer is recommended, together with a clean-up step. Cocaine and major metabolites such as ecgonine methyl ester, benzoylecgonine, and cocaethylene were shown to be present in bile in levels 3–6-times higher than in blood (Agarwal and Lemos 1996). In several cases, although the XB was not detectable in blood, it could be identified in bile (Vanbinst et al. 2002). For these reasons, there is a much smaller probability for a XB to go undetected if bile is analyzed in addition to blood samples.
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Forensic toxicology 385 Bile had been used in cases of chronic heavy metal poisoning or when XBs such as morphine, chlorpromazine, or colchicine had been implicated (Drummer and Gerostamoulos 2002). A summary of the distribution of benzodiazepines, antidepressants, cocaine, and some miscellaneous XBs in bile, blood, and liver specimens was previously provided by Agarwal and Lemos (1996) and Vanbinst et al. (2002).
fact that it is often more difficult to collect than blood postmortem. In spite of these limitations, Engelhart and Jenkins (2007), in an excellent experimental work and revision of literature, stated some conclusions:
Cerebrospinal fluid and vitreous humor Cerebrospinal fluid and vitreous humor are aqueous saline solutions, transparent, clear, and free of clotted material. They are less subject to contamination and bacterial invasion by virtue of their protected environment inside the brain, the spinal column, or the eyes. Cerebrospinal fluid is thought to be closer to the site of action of several XBs than blood, and are useful for screening analysis (Maurer 1999). Both cerebrospinal fluid and vitreous humor also contain very little enzymes and proteins. Therefore, XBs, which are highly protein bound or those that are lipophilic, tend to be found in lower concentrations in these fluids than in blood (Forrest 1993).
c. XBs concentrations are generally higher in blood than in cerebrospinal fluid;
Cerebrospinal fluid Cerebrospinal fluid is formed by the choroid plexus, a specialized tissue located within ventricular cavities of the brain (Redzic et al. 2005). Cerebrospinal fluid has a total volume of ~ 100–160 mL and is produced at a rate of 25–40 mL/h, resulting in a turnover time of 3–6 h. The protein content of cerebrospinal fluid is low, and estimated to be 0.3–0.6% that of plasma. XBs may gain entry to the cerebrospinal fluid directly through the choroid plexus, which acts as a blood–cerebrospinal fluid barrier. XBs may also enter the cerebrospinal fluid indirectly by passage across the blood–brain barrier, followed by transport from the interstitial fluid to the cerebrospinal fluid (Shen et al. 2004). Suboccipital puncture is favored to collect cerebrospinal fluid post-mortem. Alternatively, it may be aspirated from the ventricles after the skull has been removed. Normal cerebrospinal fluid should be clear, colorless, and free of clotted material. The scarce database of reference values that exists for cerebrospinal fluid restricts its applicability for the interpretation of the analytical findings. Other drawbacks lie mainly in the
a. XBs are detectable in cerebrospinal fluid; b. Concentrations measured are within the analytical capability of current testing technologies;
d. No apparent correlation exists between blood and cerebrospinal fluid for XB concentrations; e. Cerebrospinal fluid XB concentrations should not be used to estimate blood concentrations; and f. Cerebrospinal fluid /blood ratio should not be used in isolation to differentiate XB intoxication from an incidental finding. Vitreous humor The vitreous humor is located between the lens and the retina and fills the center of the eye. The vitreous humor is filled with a transparent, delicate connective tissue gel called the gel vitreous or a transparent liquid called the liquid vitreous (Jenkins 2008). The gel vitreous is a collagen gel that is water-insoluble and liquefies with age such that the adult eye contains only liquid vitreous. In Forensic Toxicology, the gel vitreous and the liquid vitreous are considered one specimen, the vitreous humor. The vitreous humor consists mainly of 99% water (as a result, the specimen is easy to work), collagen is the major structural protein and hyaluronic acid is the mucopolysaccharide (Jenkins 2008). Vitreous humor is obtained by direct gentle aspiration from each eye using a 5–10-mL syringe and a 20-gauge needle. The needle should be inserted through the outer corner (just above the junction between the upper and lower eyelids), until its tip is placed centrally in the globe (Figure 9). Vacuum tubes and heavy suction should be avoided to prevent specimen contamination with retinal fragments and other tissues (Karch 2008). It constitutes 80% of the eye and with proper techniques, 2–3 mL of fluid can be removed from each eye in an adult, while up to ~ 1 mL of specimen may be removed 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 5–10- mL syringe. The needle should be inserted through the outer corner (5 mm lateral to the limbus (corneo-scleral junction)), until its tip is placed centrally in the globe.
386 R. J. Dinis-Oliveira et al. been collected from the eye, an appropriate amount of saline can be injected back into the eye in order to reproduce the cosmetic integrity of the eye. Fluoride preservation is recommended for this specimen, particularly for ethanol quantification in diabetes-related deaths (Skopp 2004). Specimens obtained from both eyes are usually combined in one properly labelled specimen container, but different opinions on this procedure exist (Karch 2008). Interpretation of vitreous XB concentrations is difficult for several reasons: a. Very few studies have been published that relate blood concentrations to those in vitreous humor;
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b. Large ad hoc data on vitreous XB concentrations is fragmented in innumerable case reports (De Letter et al. 2000; 2002; Osuna et al. 2000; Scott and Oliver 2001; Hardin 2002; Elliott 2004; Skopp 2004; Teixeira et al. 2004; Duer et al. 2006); and c. Equilibrium between the blood and vitreous is slower than among the blood and other extracellular fluids. In vitreous humor, a delay in the uptake of XBs is likely to occur, and, inversely, there seems to be a delay in the excretion process. This suggests the presence of a barrier that is called the blood–vitreous barrier (Jenkins 2008). It has been observed that vitreous XB concentrations often reflect XB blood concentrations 1–2 hours prior to death and that any XB found in the blood will be detected in the corresponding vitreous humor specimen, given analytical techniques of sufficient sensitivity (DiMaio and DiMaio 2001). In spite of these limitations there are some advantages of humor vitreous specimen to be considered: a. A large number of XBs, including barbiturates, methanol, cocaine, morphine, tricyclic antidepressants, digitalis-glycosides, benzoylecgonine, acetaminophen, salicylate, and benzodiazepines have been analyzed (Engelhart and Jenkins 2001; Hardin 2002; Elliott 2004; Teixeira et al. 2004; Parker and McIntyre 2005; Favretto et al. 2007; Pontes et al. 2009); b. XBs that tend to be somewhat hydrophilic at physiological pH are more likely to have concentrations approaching those in blood or plasma than those XBs that are either highly protein bound (e.g. tricyclic antidepressants) or highly lipophilic (e.g. benzodiazepines). In fact, a significant negative correlation between the vitreous:blood concentration ratio and the degree of protein binding of different XBs has been reported, since XBs move in and out of the vitreous by diffusion and therefore only free XBs are able to leave the blood and enter the vitreous (Holmgren et al. 2004); c. Lacks the esterases that in blood (in vivo and in vitro) hydrolyze the metabolite of heroin, 6-acetylmorphine to morphine (Bogusz et al. 1997a; b; Guillot et al. 1997; Lin et al. 1997; Pragst et al. 1999b; Scott and Oliver 1999b;
Wyman and Bultman 2004). This justifies why 6-acetylmorphine might be present in vitreous humor, indicating heroin consumption, although blood results are negative (Pragst et al. 1999a; Scott and Oliver 1999a); d. Because the eye is remote from the central body cavity and the abdominal organs, it has been suggested that vitreous humor may be a useful fluid for the determination of XBs that are subject to post-mortem redistribution. Although that may hold true for many XBs such as digoxin, it has also been shown for others, notably cocaine and 3,4-methylenedioxymethamphetamine, an increase of the concentration in the vitreous humor after death. The authors suggested that XBs stored in the globe wall or brain had been released into vitreous humor (McKinney et al. 1995; De Letter et al. 2000); e. By virtue of its avascular and cellular nature, and protected environment inside the eye, it is less subject to contamination and early bacterial decomposition that typically occur in blood, charring, and trauma, and therefore it may be used to distinguish ante-mortem alcohol ingestion from post-mortem alcohol formation by fermentation and may provide the only opportunity to establish an ante-mortem ethanol concentration in embalmed bodies (Caplan and Levine 1990; O’Neal and Poklis 1996). Nevertheless, some studies comparing vitreous humor and blood ethanol concentrations yielded a wide variety of ratios of vitreous humor to blood ethanol (Caplan and Levine 1990). If such diversity is seen for an analyte that demonstrates minimal post-mortem redistribution effects, attempting to use vitreous humor XB concentrations to aid in interpreting heart blood XB concentrations may prove difficult (Prouty and Anderson 1990); and f. It has been shown to be particularly useful for the postmortem analysis of glucose, urea nitrogen, uric acid, creatinine, sodium, and chloride. Measuring these analytes is important for documenting diabetes, degree of hydration, electrolyte balance, the post-mortem interval, and the state of renal function prior to death (Coe 1993; James et al. 1997; Osuna et al. 1999; 2001; 2005; Tagliaro et al. 2001). Vitreous humor concentrations of sodium and chloride approximate the serum concentrations of these ions in healthy adults, especially in the early postmortem period. Potassium concentrations in the vitreous humor increase rapidly after death as potassium leaves the cells into nearby fluids (Prasad et al. 2003). Vitreous humor calcium concentrations are comparable to serum calcium concentrations. Two nitrogenous compounds, urea and creatinine, are also present in concentrations similar to serum and are stable during the early postmortem period. In post-mortem cases, these endobiotics are routinely measured as indicators for the serum concentrations of these substances at death (Coe 1993). Brain XB concentration data determined in brain tissue are not hard to find in literature (Moriya and Hashimoto 1996c; 2003;
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Forensic toxicology 387 Kalasinsky et al. 2000; 2001; De Letter et al. 2002). The brain is a useful specimen for the measurement of XBs because it is the primary site of action for many lipophilic XBs (Skopp 2004). It is also a very valuable specimen for the measurement and interpretation of XBs since it is remote from the stomach and other major organs in the body and therefore it is expected not to be affected by post-mortem diffusion and redistribution. In addition, because the brain is in a protected environment, it also tends to be more resistant to post-mortem decomposition, comparatively to other tissues or blood (Sawyer and Forney 1988). Also, metabolic activity is lower in the brain than in other tissues or blood. For example, chloroform undergoes considerable biotransformation in man, and less than 0.01% of a dose can be found in urine (Fry et al. 1972), though high concentrations were observed in brain specimens from acute fatalities (Gettler and Blume 1931). Moriya and Hashimoto (1996c) demonstrated that cocaine was stable in decomposed homogenates of human brain at 20–25°C and at 37°C over an observed period of 24 h. In rabbit brain specimens, cocaine degraded much more slowly than in blood or liver over a period of 5 days (Moriya and Hashimoto 1996c). These factors increase the likelihood of XB detection in brain specimens compared with other tissues, especially for XBs with low stability (e.g. cocaine). However, in most case reports, it was not specified which anatomic region of the brain tissue was collected for analysis, and current data establish that concentrations may vary significantly from one region to another. In an olanzapine-related death, concentrations ranged from 0.17 in the left frontal cortex to 0.86 mg/Kg in the midbrain (Merrick et al. 2001; Horak and Jenkins 2005). Therefore, the brain cannot be regarded as a single pharmacokinetic compartment, which is in accordance to its complex structure (e.g. lipid contents are higher in white matter than in gray matter). Until now no guidance exists stating that one part should be collected over another. Thus, the area of the brain from which the specimen is taken must be carefully documented. Gastric contents: Vomitus, gastric aspirate, and lavage fluid Ingestion is the preferred route of intoxication (Klaassen 2008). Oral overdoses, whether by accident or by intent, may be readily discovered through the analysis of gastric content (i.e. vomitus, gastric aspirate, and lavage fluid, or obtained during autopsy), especially if information on the XB used is not available and when specimens are obtained soon after the intoxication. Sampling of gastric content is usually performed during autopsy. The pathologist should tie off the stomach ends before removing it in block from the abdominal cavity (Figure 10). The stomach should be opened away from other specimens and tissues to avoid contamination of other viscera (Karch 2008). Because gastric content is not homogeneous, and since the total volume of gastric fluid is important for the interpretation of positive findings, the entire contents of the stomach, without preservative, should be collected and submitted to the toxicology laboratory for mixing before
aliquoting. If this is not possible, then the total volume present must be noted and provided with a representative aliquot (~ 30 mL) to the laboratory to allow the calculation of the total amount of the analyte present in the stomach. The total amount of a XB remaining in the gastric content is more important than its concentration. An estimation of the amount of XB present in the gastric volume is helpful to decide whether an analytical finding is rather more consistent with an overdose or a therapeutic dosage taken just prior to death (Skopp 2004). Gastric content can replace urine in toxicological screening, namely for those XBs that are difficult to detect in blood or urine. However, as with urine, quantitative analyses serve no purpose, since gastric levels do not reflect the amount of XB absorbed (Jickells and Negrusz 2008). Moreover, gastric content is useful to determine the time since XB intoxication and to distinguish oral from other routes of administration. A large quantity of the parent XB in the gastric content, relative to a prescription dose, is indicative of an oral overdose when supported by blood and/or tissue findings (Wetli and Mittlemann 1981; Bar-Or and Wahby 1982; McCarron and Wood 1983; Joynt and Mikhael 1985; Hassanian-Moghaddam and Abolmasoumi 2007; Schaper et al. 2007; Takekawa et al. 2007; Sengupta and Page 2008). Nevertheless, a low absolute amount present in the stomach content does not rule out the possibility of an overdose (Skopp 2004). Numerous cases have shown that it may take several hours for an individual to die from an intentional overdose, depending on the XB ingested, the amounts, co-ingestion of alcohol, general state of health, and age. It is not unusual for victims to die from an oral overdose with less than a single therapeutic dose remaining in the stomach, notwithstanding the fact that an overdose of XB can irritate the stomach lining and therefore delay gastric emptying. Extensive vomiting before death can also reduce the amount of XB remaining in the stomach at the time death (Karch 2008). In addition, the toxicologist is cautioned that low gastric content of a XB does not necessarily mean that the XB was recently consumed, or even prove that the XB was taken orally. This may represent passive diffusion (re-excretion into the gastric contents through gastric fluid) and/or ion trapping from the blood back into the stomach contents via the gastric juice, a phenomenon frequently observed, especially in XBs and its metabolites that are weakly basic in nature. The same re-excretion phenomenon can be seen with XB metabolites where, invariably, concentrations can be found in the gastric content. In addition, small quantities of XB can derive from bile, especially during agonal processes when vomiting of bile can occur (Karch 2008). Conversely, the presence of ‘ghost’ tablets in gastric content has been reported for at least one type of slow-release analgesic, where overdose or abuse was not suspected. Apparently, the wax-resin matrix of these sustained release tablets may remain in the gastric content long after the XB has diffused out (Anderson et al. 2002). Remains of undegraded tablets, capsules, or other materials of exogenous evidence, usually present if specimens are collected soon after intoxication, should be transferred to a separate container. Large amounts of capsules or tablets
388 R. J. Dinis-Oliveira et al. B
C
D
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A
E
Figure 10. To collect gastric contents, the pathologist should tie off the stomach ends (a–c) before removing it from the organ block (d). The entire contents of the stomach should be collected and mixed before aliquoting (e).
may form a gelatinous mass, which is not readily dissolved or broken up, and therefore can be found in the stomach many hours, or even a day or two, after an overdose; they are called bezoars (Ku 1996). They occur, at least in part, because gastric emptying time is delayed significantly by irritants, including large amounts of undisolved XBs residues. However, the phenomenon is also occasionally seen in patients where overdose is extremely unlikely (e.g. controlled setting such as a hospital or nursing home). This is more feasible to occur when enteric-coated tablets are involved, which do not dissolve in the stomach, but may stick together to form a small mass of tablets (Karch 2008). It is also more likely to happen in elderly victims or in other patients where gastric motility is slow. The local poisons information service or pharmacy will normally have access to publications or other aids to the identification of legitimate and sometimes illicit tablets/
capsules by weight, markings, colour, shape, and possibly other physical characteristics. Pesticides or solvents may readily be discovered due to their peculiar odor or conspicuous appearance. The odor of gastric content can potentially point to a specific XB that might otherwise elude routine detection in the toxicology laboratory. Cyanide ingestions produce stomach contents with the odor of bitter or burned almonds (Karch 2008). Although not everyone is able to discern this odor, its presence is almost certainly indicative of cyanide intoxication, and may be potentially hazardous in close quarters. In addition, great care is needed if cyanide salts or phosphides, for example aluminium phosphide, are thought to have been ingested, particularly on an empty stomach, because highly toxic hydrogen cyanide or phosphine gas may be released due to reaction with stomach acid. Additionally, the presence of these and
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Forensic toxicology 389 other volatile materials can lead to cross-contamination of other biological specimens unless due precautions are taken. Other characteristic odors include the ‘fruity-like’ odor of ethanol and its congeners, the odor of airplane glue (xylene, toluene), cleaning fluid (halogenated hydrocarbons), carrots (ethchlorvynol), and garlic (organophosphate insecticides). In many cases in which death followed the ingestion of nitrite, the agent could be identified in gastric content but not in blood (Blunt 1976). In poisonings involving heavy metals such as arsenic, mercury, or lead, specimens of stomach or the small intestine content should also be taken for toxicological analysis. In addition, illicit XBs are frequently smuggled by ingestion of balloons or condoms filled with it (the body-packer syndrome). If these devices burst and an acute XB-mediated death occurs, evidence of these items may be seen in the gastric content at autopsy (Hassanian-Moghaddam and Abolmasoumi 2007; Kelly et al. 2007; Veyrie et al. 2008; DinisOliveira et al. 2009b). If intestinal content is sampled, the anatomical source from intestinal loops should be notified. Sweat Sweat is a fluid excreted from the sweat glands (eccrine and apocrine types). The eccrine glands are widely distributed at the surface of the whole body while the apocrine glands are located in the axillary, mammary, genital, and perianal regions. The maximal excretion volume was reported to be ~ 2 L/day in healthy subjects and ~4 L/day in trained sport athletes (Suzuki and Watanabe 2005), but the volumes and constituents are greatly different according to individuals, types of gland, and various stresses (emotional, physical, and thermal) (Sunshine and Sutliff 1996). The sweat analysis started in ~ 1970, and showed that various XBs can be detected in sweat (Sunshine and Sutliff 1996). Johnson and Maibach (1971) reported that there is a close relationship between pKa of a XB and its amount of excretion into sweat, and also between XB concentrations in sweat and in plasma. However, collecting sweat is not easy and it is difficult (or even impossible) to obtain it quantitatively. Therefore, sweat should be applied for qualitative toxicological analysis. In the majority of cases, the sweat is collected by wiping the skin surface with cotton, gauze, or towel and by using patches attached to the skin (Sunshine and Sutliff 1996). Sweat collection is non-invasive and commercially available sweat patches may be worn for an extended period of time (10–14 days or so). Controlled dose studies concluded that a sweat patch must be worn for a minimum of 24 h to collect sufficient amount of XB for analysis (Cone et al. 1994), which itself represents a limitation. The longer periods of XB excretion into sweat allied to the possibility of an extended use of collection patch, enables XBs detection even 1–4 weeks after single administration (Inoue et al. 1995). The sweat patch can be worn on the upper arm (the most common area), lower rib cage area, and the upper back (Jenkins 2008). Due to the aggressive nature of the adhesive, as a general rule, care should be taken to ensure that the application area is not subject to vast
amounts of flexing. Prior to application of the sweat patch, the skin should be cleaned using two isopropanol wipes, allowing alcohol to evaporate completely, otherwise skin irritation can develop due to isopropanol trapped beneath the sweat patch (Jenkins 2008). The components absorbed could be eluted with water, followed by extraction of XBs before instrumental analysis. Amniotic fluid The increasing use of XBs by expectant mothers has led to an increased need for pre-natal toxicological testing. For instance, exposure to drugs-of-abuse may result in higher rates of congenital anomalies and neonatal complications. Therefore, identification of gestational XB exposure may benefit the newborn in terms of increased vigilance and monitoring of the infant by medical and social services (Jenkins 2008). Amniotic fluid consists of a filtrate of maternal blood that surrounds and protects the embryo during pregnancy. It is composed of 99% water and contains cells and fat that may give the liquid a slightly cloudy appearance. Due to its high water content, little analytical interference exists, making this specimen easily applied to routinely used toxicological tests. At the end of 9 months of gestation, ~ 1.5 L of amniotic fluid are normally present. Amniotic fluid acts as a fetal excretion reservoir, accumulating XBs throughout gestation. Since amniotic fluid is constantly in circulation (being swallowed by the fetus, processed, absorbed, and excreted by the fetal kidneys as urine at rates as high as 50 mL per hour) and since the fetus is encapsulated, prolonged exposure to XBs may ensue. XBs can enter amniotic fluid by passive diffusion across the placenta and from excretion of fetal urine in the latter stages of gestation (Gray and Huestis 2007). Another possible route of exposure via amniotic fluid is transdermal diffusion, early in pregnancy when the skin is poorly developed and late in pregnancy when the production of vernix caseosa takes place. The major limitation of amniotic fluid is sample collection. In fact, this sample can only be non-invasively collected at birth or as excess specimen from amniocentesis (typically 5–30 mL of amniotic fluid is removed) that usually takes place between the 16th and 20th week of pregnancy (Jenkins 2008). Positive toxicological analysis for a specific XB or their metabolites suggests that the fetus has been exposed to these substances through maternal blood circulation. Commonly, this sample is not collected for monitoring in utero XB exposure alone (Gray and Huestis 2007; Lozano et al. 2007). A maternal serum sample obtained at the same time may provide complementary toxicological data and help to assess the relative risk to the fetus. In addition, hair, nail clippings, or meconium could be useful in the interpretation of forensic results. Exhaled air Analysis of exhaled (expired) air is developing fast since it enables a non-invasive diagnostic of exposure. Measurement of concentrations of volatile XBs in exhaled air by infrared or other devices is essential in roadside testing for ethanol as evidence for prosecution of drunk drivers (Tardif et al. 2004;
390 R. J. Dinis-Oliveira et al.
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Turner et al. 2006; Kamat et al. 2007; Tardif, 2007). Breathtesting eliminates the need for taking blood samples, can be used at the roadside, and the result is immediately available, making it possible for police officers to charge the suspect on the scene without delay. The ethanol metabolite, acetaldehyde, has also been measured in this specimen. Exhaled air analysis is also valuable in assessing exposure to other XBs such as carbon monoxide (Paredi et al. 1999; 2000), hydrogen cyanide (Stamyr et al. 2008), and anesthetics (Perl et al. 2009). Obviously the use of this sample is not possible post-mortem, due to the need to take breath directly from living subjects (Harrison et al. 2003). Saliva/oral fluid Since the report in the middle 1950s that XBs were movable from blood to saliva, many researchers examined the usefulness of saliva analysis, and clarified that XB concentrations in saliva reflected those in blood. Saliva is the excretion product originating from three pairs of major salivary glands (parotid, submandibular, and sublingual), a great number of minor salivary glands, the oral mucosa, and gingival crevices. Small amounts of cellular debris may also be present. As this excretion product is actually a fluid mixture, the term ‘oral fluid’ seems more appropriate, instead of ‘saliva’ or ‘whole saliva’ (Malamud and Tabak 1993). The New York Academy of Sciences meeting on saliva testing, in 1993, agreed to use the word saliva for glandular secretions collected directly from the saliva glands (most often the parotid glands), and oral fluid for fluid collected by placing absorbants in the oral cavity or by expectoration (Malamud and Tabak 1993). Although saliva may be collected from, for example, the parotid gland by cannulation of the glandular duct, the collection of mixed whole saliva is normally the only practical alternative. A variety of methods are available for oral fluid collection, including spitting, draining, suction, and collection on various types of absorbent swabs (Drummer 2006; Gallardo and Queiroz 2008). Several devices are also commercially available for on-site analysis, including instruments that provide an electronic readout and hand-held cartridges that require visual identification. The main advantage of these devices is that they provide a preliminary XB result within minutes without the need for sophisticated laboratory screening equipment. The results provided by these devices must be confirmed in the laboratory. Subjects should not brush their teeth or practice other methods of oral hygiene for several hours before saliva is collected. Water corresponds to 99% of the oral fluid content. Other components such as proteins (0.3% mucins and 0.3% digestion enzymes, largely amylase) and mineral salts are also present. Its pH is 6.8 in resting situations, but an increase in the salivary flow turns it more basic (approaching the plasma’s pH) as a result of higher osmolarity (Kintz and Samyn 2000). All these characteristics are influenced by a variety of factors, such as the circadian rhythm, the type of the salivation stimulus, hormonal changes, stress, and therapeutic drugs (Aps and Martens, 2005). The total volume of oral fluid produced by an adult may be 1000 mL/day, with typical flows
of 0.05 mL/min while sleeping, 0.5 mL/min while spitting, and 1–3 mL/min or more while chewing (Crouch 2005). Use of acid lemon drops or a few drops of 0.5 mol L−1 citric acid are amongst chemical stratagems adopted to stimulate salivary flow, which can result in changes in salivary pH that can alter the secretion rate of ionizable XBs. Using paraffin wax and Parafilm to stimulate oral fluid production may absorb highly lipophilic XBs. The oral fluid should be allowed to accumulate in the mouth until the desire to swallow occurs before being expelled into the collection vessel. Special attention must be given if secretion of oral fluid was subjected to stimulation or not. When the fluid enters the mouth, carbon dioxide is lost and the pH increases. Dawes and Jenkins (1964) demonstrated that oral fluid pH is inversely proportional to flow rate and the reabsorption of sodium in the salivary tubules. At faster flow rates, less sodium is reabsorbed in the tubules on the way from the saliva glands to the saliva outlets in the mouth and the pH rises. For this reason, unstimulated oral fluid has a low pH (at low flow rates between 6.0–7.0, and fairly constant) and stimulated oral fluid has a higher pH (it can reach as high as 8.0). When the fluid reaches the mouth it is hypotonic to plasma. Like the liver, kidney, and brain the salivary glands, are well supplied with arterial blood. Salivary glands are activated by the autonomic nerves. Generally, sympathetic stimulation via noradrenaline causes low levels of fluid and high concentrations of protein, while parasympathetic stimulation via acetylcholine induces large amounts of fluid secretion. Human saliva normally has a lower pH than human plasma. Therefore, the oral-fluid:plasma ratios for acid XBs are generally less than unity while ratios for basic XBs are greater than unity, increasing levels of basic XBs in oral fluid. For XBs that have a pKa between 5.5–8.5, the oral fluid:plasma ratio can vary between stimulated and unstimulated oral fluid. This is true for many drugs of abuse. For this reason, it is more conservative to use a cut-off value for drugs of abuse in oral fluid rather than to determine the absolute concentration. The most common example given is that of cocaine, which has a pKa of 8.6 (Schramm et al. 1992). The oral fluid:plasma ratios for cocaine can be as higher as 6, when the oral fluid pH varies from 6.5 -8.5. Different mechanisms of XBs transport are thought to occur, such as passive diffusion through the membrane, active processes against a concentration gradient, filtration through pores in the membrane and pinocytosis (Gallardo and Queiroz 2008). Most of the XBs enter oral fluid by a mechanism of passive diffusion, which is dependent of the molecular weight (a molecular weight of less than 500 Da favors diffusion), liposolubility, pH and pKa, protein binding, and ionization state (Paxton 1979; Aps and Martens 2005) of the XB, as shown by the Henderson–Hasselbach (Spihler 2004) equation. Diffusion requires that the XBs (or metabolites) be lipid-soluble, non-ionized, and unbound. Therefore, the concentrations of XBs in oral fluid represent the free non-ionized fraction in the blood. Since these are the forms of the XBs that cross the blood–brain barrier and affect performance and behavior, oral fluid is a good specimen for
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Forensic toxicology 391 detecting patient compliance with medication, XB involvement in driving behavior, fitness for duty, or impairment of performance for many XBs. However, efforts to use oral fluid concentrations to predict blood free-XB concentrations or degree of performance impairment in individuals have not reached the accuracy of blood measurements. Without knowing the instantaneous oral-fluid pH, oral-fluid XB concentrations may not be extrapolated to give blood XB concentrations. The protein binding of XBs in plasma is mainly to albumin. Oral-fluid mucoproteins have very little binding capacity for XBs, although oral fluids may contain albumin from the gingival crevicular fluid. The advantages of oral-fluid XB testing are various. In principle, oral-fluid XB concentrations can be related to plasma free-XB concentrations and therefore to the pharmacological or toxicological effects of XB. Second, oralfluid collection is non-invasive, easier than venepuncture, can be done on-site under direct supervision without loss of privacy and can be done by the donors themselves in most situations (Spihler 2004). In consequence, the risk of an invalid specimen being provided or sample adulteration and/or substitution (which are likely to occur in urine analysis) is reduced. In addition, oral fluid monitoring may be especially important when multiple serial samples are needed or when XB concentrations in children are required (Kim et al. 2002). The feasibility of detecting XBs in oral fluid samples obtained from impaired drivers was first investigated by Peel et al. (1984). Authors found that the presence of XBs in oral fluid correlated well with officer judgments of driving while intoxicated. This was confirmed in a comparison of saliva testing to urinalysis in an arrestee population (Yacoubian et al. 2001) and in drugged drivers (Steinmeyer et al. 2001). Several methods have been described to detect opiates, cannabinoids, amphetamines, cocaine, benzodiazepines, ketamine, γ–hydroxybutyric acid, antibiotics, analgesics, cyanides and other tobacco compounds, sildenafil and many other XBs (Gallardo and Queiroz 2008). However, like any fluid from human subjects, oral fluid may transmit infectious agents and should be handled with the appropriate universal precautions for human biological fluids. Oral fluid contains mucopolysaccharides and mucoproteins that make it less fluid (more viscous) and less easily poured or pipetted than urine or plasma. Routine dilution with aqueous collection buffer is advocated by some authors to minimize this problem. Some XBs, medical conditions (when the donor is unconscious or sedated), or anxiety can inhibit oral-fluid production and cause dry mouth. Therefore, oral-fluid may not be available from all individuals at all times or they can produce insufficient amounts of sample for analysis. In addition, reliable oralfluid collection requires a co-operative individual and even then is not without problems. Finally, because oral fluid XB concentrations depend on plasma XB concentrations, XBs that have a short plasma half-life and are cleared rapidly from the body are detectable in oral fluid for a short time only (Spihler 2004). This is a potential limitation over the
detection of XBs in the hair, sweat, or urine. In general, XBs are detectable in the plasma and oral fluid from the time that the XB enters the general circulation until approximately four half-lives after exposure. Another limitation drives from the fact that the majority of the XBs exposure are from the oral route. Ingested XBs as well as those that can be smoked or snorted may be detected in high concentrations in oral fluid following recent use, due to residual amounts of XBs remaining in the oral cavity. Therefore, the analytical results are not accurate since XBs concentrations found in the oral fluid do not reflect their blood concentration. In cases that involve these routes of administration, 2–4 h should elapse. Jenkins et al. (1995) showed that the oral fluid:plasma ratio for smoked heroin was 100–400-times higher than when heroin was administered intravenously. After smoking heroin, heroin was detected in oral fluid for up to 24 h, compared to up to 30 min after heroin was intravenously administered. The same authors reported that after smoking cocaine the oral fluid:plasma ratio was 300–500-times higher than that found after cocaine was administered intravenously. The pyrolysis product of cocaine, anhydroecgonine methyl ester, was detected in oral fluid collected after smoking cocaine, but not in plasma. Similarly, cannabinoids found in oral fluid almost totally result from oral deposition of cannabinoids from smoked marijuana rather from secretions or diffusion into oral fluid (Ohlsson et al. 1986). O’Neal et al. (2000) reported codeine oral fluid:plasma ratios of 75–2580 in the first 15–30 min after dosing and of 13–344 for several hours after oral administration of liquid codeine phosphate, despite decontamination efforts by having the subjects brush their teeth and vigorously rinse their mouth prior to oral fluid collection. The formation of oral mucosa depots of XBs, which are rapidly absorbed into the blood circulation, is used for XB administration. Sublingual or buccal absorption of XBs, such as nitroglycerine, buprenorphine, or fentanyl, has the advantage of very rapid delivery that bypasses the liver and gastrointestinal first-pass metabolism. XBs administered by this route also produce large concentrations of the parent XB in oral fluid, but with a short detection window, since XBs are rapidly absorbed (Spihler 2004). Feces After the kidney, the second most important route of elimination of many XBs is through feces. Nevertheless, fecal excretion of XBs is a complex process that is not as well understood as urinary excretion (Klaassen 2008). The main mechanisms contributing to the presence of XBs in feces are the following: a. Incomplete absorption, as occur in paraquat intoxications due to its hydrophilic nature (Dinis-Oliveira et al. 2008); b. Intestinal secretion, which likely occurs by passive diffusion out of enterocytes or via exfoliation of intestinal cells during the normal turnover of this epithelium; and c. Biliary excretion—perhaps the most significant source contributing to the fecal excretion of XBs, and is even
392 R. J. Dinis-Oliveira et al. more important for the excretion of metabolites. P-glycoprotein is considered the main transporter involved, as previously proved for paraquat (DinisOliveira et al. 2006b).
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The analysis of feces is rarely performed in clinical chemistry, analysis being restricted mainly for pharmacokinetic and metabolism studies (Sorg et al. 2009) or if the question of ante-mortem XB leakage from ingested packets is raised. Unlike plasma, urine, and other fluid samples, feces are not homogeneous, and thus it is often necessary to analyze the whole sample or homogenize the whole sample and prove that the fraction taken for analysis is representative of the whole (Flanagan et al. 2007). Meconium Prenatal exposure to XBs is an ongoing concern, with significant impact on neonatal health and development (Meeker and Reynolds 1990; Dusick et al. 1993; Gingras et al. 2004; Wouldes et al. 2004; Hurd et al. 2005; Smith et al. 2006). Meconium, the first fecal matter passed by a neonate, has recently been given much attention because it is a useful specimen to determine fetal exposure to XBs, it is easily collected and non-invasive, large amount of sample can be collected and can provide information regarding long-term exposure. Meconium, in comparison to urine, is easily collected. For newborn urine collection, bags are taped to the babies, and these often become detached or urine is spilled. Meconium is simply scraped from a diaper and placed in the collection vial (Jenkins 2008). Post-mortem meconium analysis can also constitute additional help in the medico-legal practice, in cases of sudden infant death syndrome and of unexplained late fetal death. It is identified most commonly by its dark green/black color because of the presence of bile and a lack of the odor of regular feces. It is a highly complex matrix consisting of water, mucopolysaccharides, bile salts, bile acids, lanugo (fine neonatal hair), desquamated epithelial cells from the gastrointestinal tract and skin, lipids, proteins, as well as the residue of swallowed amniotic fluid (Chan et al. 2004a; b; Gareri et al. 2006). Fetal swallowing is thought to be the mechanism by which XBs are concentrated in the meconium, since as the fetus releases urine into the amniotic fluid, any excreted XBs and metabolites are then swallowed and ultimately deposited into the meconium (Browne et al. 1992; Ostrea et al. 2006). In addition to this mechanism, fetal exposure is a product of maternal consumption, metabolism and elimination, placental transfer and metabolism, and also fetal metabolism (Gallardo and Queiroz 2008). The timing of meconium formation has been variably reported in the literature. The assertion that meconium begins to form at ~ 12 weeks of gestation is likely to be the most accurate, since it is at this time that fetal swallowing of amniotic fluid begins (Kwong and Ryan 1997), and the formation of meconium has been evidenced at this time period by the presence of cocaine found in the meconium of early gestational fetuses (Ostrea et al. 1994). XBs concentrations in
meconium are generally higher than in urine because of its accumulation over several months of gestation (Ostrea 2001; Ostrea et al. 2001). One major advantage of meconium is a relatively wide window for sample collection. Unlike urine, which allows the detection of fetal XB exposure for only 2–3 days before birth, meconium extends this window to ~ 20 weeks pre-partum (Moore et al. 1998; Kintz and Samyn 2000), and therefore history of in utero XB exposure during the second and third trimesters is highly possible. These factors make meconium an optimal matrix for identifying in utero exposure as it is considered a preserved record of the ultimate exposure by the fetus. After birth, meconium is excreted by the neonate several times a day for the first 1–5 days postpartum. Viable analysis appears to be optimal via collection within 72 h, since in the later stages of meconium excretion a matrix of meconium and feces is produced. In extremely low birth weight infants, which is of particular interest in the XB exposed neonatal population, median age of first stool is 3 days, with 90% of infants passing their first stool by day 12 (Verma and Dhanireddy 1993). For these reasons, the possibility of sample collection beyond 48 h post-natally is a remarkable advantage of this matrix (Ostrea 2001; Ostrea et al. 2008). Issues relating to the screening and confirmation of most drugs of abuse in meconium have been reviewed (Lewis et al. 1994; Moore et al. 1995a; b; 1998; Le et al. 2005; Lopez et al. 2007; Gallardo and Queiroz 2008). Most post-mortem toxicology laboratories are not currently performing meconium analysis. While potentially useful, there are several limitations that must be considered. Because meconium forms layers in the intestine as it is being deposited, it is not a homogeneous specimen (Karch 2008). As with other non-homogeneous specimens, such as gastric content, it is important that all available specimens are collected and thoroughly mixed before aliquoting. Meconium analysis is more labor intensive and requires more time to analyze than urine. Often the sample size obtained is small, and the concentration of XBs found in meconium is relatively low (Jenkins 2008). However, advances in technology have improved the sensitivity of testing procedures, so this is increasingly of less importance. Liver The liver has been ranked as the primary solid tissue for use in post-mortem toxicology, and often the XB analysis, resulting from this tissue, complements the blood toxicology data (Luckenbill et al. 2008; Gronewold et al. 2009; Vance and McIntyre 2009). For many XBs, especially those with chemically ‘basic’ character (e.g. alkaloids), higher concentrations can be found in the liver comparatively to blood (Jenkins 2008). Most case reports illustrating toxicology findings include concentrations of the XBs in liver. It is also the favored specimen when blood is not available due to exsanguination, fire, or decomposition. As a specimen, liver also has the advantages that it is the major metabolic organ, sufficient quantities are available for analysis, and it is reasonably homogeneous and relatively unaffected by postmortem redistribution or post-mortem diffusion compared
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Forensic toxicology 393 with blood. Nevertheless, XBs concentrations in the lobe proximal to the stomach may artificially increase by postmortem diffusion in cases of oral overdose. Therefore, use of tissue from deep within the right liver lobe is preferred (Figure 11) (Pounder and Smith 1995; Pounder et al. 1996b). Bile should be collected prior to the liver specimen, to prevent contamination. Liver has been found to be particularly suitable to determine tricyclic antidepressants and many other XBs that are highly protein bound (Apple and Bandt 1988; Apple 1989; Moriya and Hashimoto 1999c; Davis et al. 2001). It is useful for the phenothiazine neuroleptics, which have a large dosage range (Robinson et al. 1974; Apple and Bandt 1988; Anderson et al. 1999; Davis et al. 2001; Yarema and Becker 2005; Kirkton and McIntyre 2006; Wenzel et al. 2006). Analysis of a liver tissue specimen may help to differentiate acute overdose from therapeutic use of XBs with a narrow dosing window. In cases where the concentration of basic XBs in blood is high and ratios of liver-to-blood XBs concentration exceed 10, a XB fatality is strongly suggested if no other interceding cause of death is present (Karch 2008). Smaller ratios, even with high heart blood concentrations, tend to suggest a greater potential for post-mortem redistribution of XBs into the blood. Due to the lipid-soluble nature of many XBs and the high lipid content of the liver, long-term sequestration for many XBs in this organ is likely to occur, originating with a longer half-life than the traditional half-life in circulating blood, and this should be taken into account when interpreting very low concentrations of XBs in the liver (Jenkins 2008). Limitations of utilizing liver include the necessity to produce a homogenate prior to extraction, matrix effects observed with routine analytical techniques, and lack of a database to aid the interpretation of XB concentrations.
Lung High concentrations of XBs are frequently found in lung tissue, especially after poisoning by intravenous or inhalatory routes. Depending on the properties of a XB, concentrations in lung tissue can be higher than in liver (Dinis-Oliveira et al. 2008; 2009a). When solvent abuse or an anestheticmediated death is suspected, lung and brain specimens must be available for toxicological investigation. In addition, air may be collected directly from the trachea with a syringe and injected into a sealed vial to be used for headspace analysis. In paraquat intoxication-related deaths, lung should always be analysed (Dinis-Oliveira et al. 2006a; 2008). Paraquat mainly accumulates in the lung (pulmonary concentrations can be 6–10-times higher than those in the plasma), where it is retained even when blood levels start to decrease. Accumulation occurs against a concentration gradient, through the highly developed polyamine uptake system. A negative result in blood is particularly common, lung being soaked in paraquat (Dinis-Oliveira et al. 2006a; 2008). Pounder et al. (1996b) observed that amitriptyline and paracetamol added to the stomach of XB-free cadavers diffuse into the left lung, left lobe of the liver, the spleen, and into the pericardial fluid. To reduce the problem of post-mortem XB diffusion from the stomach and gastrointestinal tract, it has been recommended that lung should be sampled from the apex rather than the base (Figure 12) (Pounder et al. 1996a). Kidney A kidney specimen can be a useful sample for XB identification since most XBs and metabolites are excreted into urine and therefore will pass through the kidneys. It is particularly important in cases of heavy metal poisonings due 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 (a–d; dashed line).
394 R. J. Dinis-Oliveira et al. Triunfante et al. 2009). In addition, structural damage to the kidney due to heavy metal or ethylene glycol exposure may be documented histologically, supporting the toxicological analysis (Post et al. 1984; Scott et al. 1987; Sugita and Tsuchiya 1995; Barregard et al. 1999; Debacker et al. 2000; Rumbeiha et al. 2000; Alonso et al. 2005; Ferrari and Giannuzzi 2005; Takahashi et al. 2008).
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Spleen Spleen, an organ rich in blood, is useful for the analysis of XBs that bind to hemoglobin, such as carbon monoxide and cyanide. Frequently, in fire deaths where extensive charring is present, spleen may be the only useful specimen available to perform these assays. Cardiac muscle A cardiac muscle specimen can also be a useful sample for XB identification. Although no specific roles exist to obtain heart muscle, collection of samples from the left ventricle has been reported by several authors (Figure 13) (Hikiji et al. 2008; Luckenbill et al. 2008; Thiblin et al. 2009). Skeletal muscle Skeletal muscle has several advantages and many potential applications in post-mortem Forensic Toxicology. It meets many of the criteria of an ideal forensic specimen: a. It is present in large quantities (often represents the greatest single mass of XB in a body and will therefore represent a greater body burden of XB than any other tissue mass, ~ 30 Kg, or 43%, of a 70-Kg person); b. Almost always available even in putrefaction, trauma, and burning; c. Less affected by decomposition than blood and internal organs and therefore it may be useful as an indicator of post-mortem blood concentrations (Garriott 1983; 1991; Christensen et al. 1985). Surprisingly, even cocaine, which is known to be unstable in blood, has been iden-
Figure 12. Lung should be sampled from the apex rather than the base of the right lung (dashed line).
tified in numerous cases of decomposed, dried skeletal muscle (Manhoff et al. 1991); d. Sampling is possible from various sites away from XB reservoirs in liver, lung, or gastric content. Normally, specimens of iliopsoas muscle (right or left of the lumbar portion of the spine) are collected (Figure 14) (Schloegl et al. 2006a; b); and e. Concerning the homogeneity of the tissue, different theories exist (Christensen et al. 1985; Garriott, 1991; Williams and Pounder 1997). Interesting data on XB concentrations from muscle specimens were provided by Langford and coworkers (Langford and Pounder 1997; Langford et al. 1998) and Williams and Pounder (1997). Twelve different muscle samples collected from overdose cases (n = 11) as well as from cases with chronic therapeutic use (n = 3) were analyzed in the first two reports. These results were compared with each other and with the XB concentration in corresponding femoral blood. The muscle-to-blood ratio was influenced by the time lapse between XB exposure and death as well as by the volume of distribution of the XB. The within-case variability observed in muscle specimens supports the opinion that XB analysis on skeletal muscle is rather qualitative than quantitative in nature. The same conclusion was provided by Williams and Pounder (1997), analyzing a series of eight fatal XB overdoses. Studies performed by Garriott (1991) showed that XB concentrations in thigh muscle reflect XB concentrations in blood for many common basic XBs and ethanol, except in cases of an acute XB death where muscle XB concentrations may be lower than blood due to inadequate time for tissue equilibration. The analysis of thigh muscle was proposed to be especially useful in cases where XBs suspected of undergoing post-mortem redistribution or diffusion are detected in the heart blood (Garriott 1991). Christensen et al. (1985) also suggested to collect the muscle extremity, where possible, as XBs concentrations in other muscles, such as abdominal 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 toxicologists to recommend skeletal muscle to be collected in all cases where XBs may be implicated in the cause of death (Christensen et al. 1985). One limitation of skeletal muscle is that its homogenization, prior to analysis, is very difficult, 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 analyzed due to the difficulty in reliably extracting XBs and because of substantial variability in parent compound/ metabolite concentrations from one site to another. However, these tissues represent the greater mass of a body and a greater
Forensic toxicology 395 A
A
B
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B
Figure 13. Collection of cardiac muscle from the left ventricle has been suggested (dashed line).
body burden of XB than any other tissue. Until now, there is no guidance from which part of the body should this sample be obtained, but abdominal subcutis adipose tissue has been considered in the majority of forensic works (Schloegl et al. 2006b). Adipose tissue acts as a reservoir for many lipophilic XBs (Sorg et al. 2009). For instance, Δ9-tetrahydrocannabinol has an avid affinity for it with concentrations ~ 200-fold higher than that in circulating blood (Chu 2002). Although few reports on XB detection in post-mortem adipose tissue have been published (Levisky et al. 2000; 2001), some important conclusions were ascertained. It was reported that XBs identified in the adipose tissues of cadavers are there because of ante-mortem deposition and not post-mortem redistribution (Levisky et al. 2000; 2001). To reach these conclusions, the authors examined tissues with and without livor mortis. Livor mortis (also referred to as hypostasis) and post-mortem lividity are terms used to describe the staining of the skin surface and internal organs by the settling of blood, under the influence of gravity, after death (Henssge et al. 2002). Livor mortis can be considered a form of vascular post-mortem redistribution involving the movement of blood within the vascular compartments. In this case, even though there is obvious redistribution of vascular components, including red cells and hemoglobin, there was no evidence of the infiltration or diffusion of XBs from vascular sources outside the adipose layer: the cocaine concentrations in the tissues with livor
Figure 14. For skeletal muscle, normally, specimens of iliopsoas muscle (right or left of the lumbar portion of the spine) are collected (dashed line).
mortis are essentially the same as those without it (Levisky et al. 2001). Also, since adipose tissue is poorly vascularized, and only ~ 2% of the blood supply is distributed throughout this layer, any contribution of XBs coming from blood vessels would be minimal. In an animal study, higher amounts of fatty ethyl esters, as post-mortem markers for ethanol intake, were present in adipose tissue, compared to liver (Salem et al. 2001; Refaai et al. 2002). As for skeletal muscle, adipose tissue may be useful for post-mortem examination of injection sites, due to higher XB concentration. Nasal, oral, and skin swabs Usually, cotton tips are used to collect nasal, oral, and skin swabs by carefully rubbing over the suspected areas (Skopp 2004). The tip may be moistened with methanol or ethanol prior to sampling. The swabs should be placed into a transport tube and sealed. Skin swabs for XB analysis should be taken from sites that had been covered by clothes to minimize external contamination (Skopp 2004). Nasal and oral swabs allow the simple detection of a XB. However, a positive finding does not prove that the XB had been intranasally administered or orally ingested because pulmonary edema fluid may be the source of the material tested. For example, the simple detection of cocaine in a nasal swab does not prove that the XB was ‘snorted’. Any fluid secreted by the body, including sweat, vaginal fluid, and nasal secretions, will contain some concentration of the XB. The same is true for skin swabs. Although
396 R. J. Dinis-Oliveira et al. skin swabs have became very popular in roadside testing in some countries, in the last decase (Skopp and Potsch 1999), this specimen is of limited value in post-mortem toxicology.
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Keratinized tissues In recent years, keratinized tissue such as nails and hair have received considerable attention, since these samples may provide retrospective information that far exceeds that from blood or urine. Hair Hair possess a shaft and root and is constituted by proteins (65–95%, mainly keratin), water (15–35%), lipids (1–9%), and minerals (0.25–0.95%) (Wennig 2000). A rich capillary system, which provides the growing hair with the necessary metabolic material, surrounds the hair follicle (Pragst and Balikova 2006). The growth rate is dependent, to some extent on individual factors, such as age, hair care, and environmental factors, anatomic region, sex, health conditions, pigmentation, and culture or race (Wennig 2000). Growth rates are ~ 0.6–1.4 cm and 0.6 cm per month for scalp and pubic hair, respectively (Kintz 2004). Beard hair is also a suitable specimen for analysis. This type of hair grows at ~ 0.27 mm per day, and therefore can be collected on a daily basis with an electric shaver. Therefore, significant differences in the anagen (active growing)/catagen (transition)/telogen (resting) stages of the hair growth cycle, and in the respective growth rate, exists (Mangin and Kintz 1993), justifying that the length, the color, an obvious cosmetic treatment, and the collection site of the specimen should be documented in order to facilitate the interpretation of the results. For instance different XB concentrations between pubic or axillary hair and scalp hair were found (Han et al. 2005) and some XBs are susceptible to degradation by cosmetic treatment and/or by sunlight (Skopp et al. 1997; 2007). Hair has a long history as a useful specimen in Forensic Toxicology, especially used as a supplementary tool to disclose or confirm previous XB use (Karch 2008). Hair is a unique material for the retrospective investigation of chronic exposure since it provides a longer window of detection compared to blood or urine, and is less invasive to collect. Long scalp hair may provide retrospective information of the previous 5–7 years (Daniel et al. 2004). In addition, analysis of hair for XBs is not more difficult or challenging than testing in other matrices, since the application of analytical methods and instrumental approaches is, in most cases, quite similar, regardless of the initial sample preparation. Hair analysis has also been successfully applied on exhumed human bodies. In some circumstances, post-mortem specimens may not be available, and hair, due to its resistance to decay, could be the only sample available for testing. Hair may also survive longer after burial than other tissues (Kintz 2004). Interpretation of positive findings can be augmented by the segmentation of the hair strands to assist in determining the time of exposure and define historical XB use or changes in XB habits (or abstinence) (Smith 1964; 1976; Kintz et al. 2006; 2007b). A decrease in XB concentration in the proximal sections of hair
may indicate attenuation of use, this finding being useful in cases in which a relapse in XB use could have been related to death. Traditionally, hair, along with fingernails, has been the specimen of choice in determining chronic heavy metal poisoning such as arsenic, mercury, and lead (Kintz et al. 2006; 2007b). Keratin, found in large amounts in hair and nails, is an excellent source of sulfhydryl groups to which heavy metals bind to form covalent complexes. Since 1979, when morphine was first detected in a hair specimen from a heroin user (Baumgartner et al. 1979), numerous other XBs have been identified in hair besides heavy metals (Uematsu 1993a; b; Kintz 2004; 2007; Kintz et al. 2007a). In drug-facilitated crimes, the detection of a particular compound, such as γ–hydroxybutyric acid (Kalasinsky et al. 2001; Goulle et al. 2003; Kintz et al. 2003; 2004; 2005; 2008), in hair, has been used to document the exposure, but usually a negative finding cannot exclude an exposure (Cheze et al. 2005). If a single exposure to a XB is suspected, as in drug-facilitated sexual assault, but the suspected XB is not detected in blood or urine, waiting for 1–2 months for head hair to grow and then performing segmental analysis may reveal the presence of the XB (Kintz et al. 2003). Other important applications are the detection of in fetal utero exposure, resulting from maternal XB use, and the use of XBs in cases of child abuse (Klein et al. 1992; 1993; Koren, 1995; Koren and Klein 1997; Koren et al. 1998; 2002; 2008; Klein and Koren, 1999; Boroda and Gray 2005). The use of hair in workplace XB testing is controversial due to issues such as environmental contamination (Wang and Cone 1995; Cone 1996; 1997; 2001), washing techniques (Blank and Kidwell 1993; 1995), sex or ethnic bias, the difficulty in performing quantitative analysis (Welch et al. 1993), and establishing cut-off concentrations (Kintz 1995; 2004; Kintz and Mangin 1995; Kintz et al. 1995). Although the precise mechanisms involved in the incorporation of XBs into hair have not been completely clarified, three processes have been proposed (Blume-Peytavi et al. 2008; Jenkins 2008; Karch, 2008): a. Passive diffusion from blood capillaries into the growing cells at the base of hair follicle; b. Diffusion from sweat or sebum secretions; and c. Passive exposure to XBs from an external source, such as from smoke or dirty hands, and secondary to dissolution of the XB into the XB-free sweat. It is virtually impossible to distinguish between the presence of XBs derived from these two latter mechanisms and that proceeding from real administration, which is explained by the fact that the XBs are in an aqueous moiety, enhancing their incorporation, and because of the fact that hair is very porous and can increase its weight up to 18% by absorbing liquids, which facilitates incorporation of XBs into the hair. This is the reason why environmental exposure is sometimes called the ‘stumbling block of hair testing’. Incorporation of XBs is also affected by the melanin content of the hair and by the substances’ lipophilicity and basicity. For instance, basic
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Forensic toxicology 397 XBs incorporation in pigmented dark hair can be ~ 10-fold higher than in non-pigmented grey hair (Pragst and Balikova 2006), a fact that has been suggested to be the consequence of XBs binding to melanin (Rollins et al. 2003; Mieczkowski and Kruger 2007). After incorporation, XBs are not further metabolized. There are some recommendations about hair specimen collection (Bost 1993; Henderson 1993; Miller et al. 1997; Harkins and Susten 2003; Cheze et al. 2004; Flanagan and Connally 2005; Flanagan et al. 2005; Madea et al. 2007). If possible, hair should be collected from the posterior vertex region of the scalp or the back of the skull, where the average hair growth rate is fairly constant (Figure 15), and the hair is less subject to age- and sex-related influences, and has been extensively studied. In addition, on the scalp of an adult, ~ 85% of the hair is in the anagen phase and the remaining 15% is in the quiescent stages (catagen and telogen) at any time phase (Henderson 1993). Cut hair is usually preferable since hair roots, often containing high amounts of XBs resulting from an acute intake, are then excluded. In cases with a suspicion of a recent poisoning, analysis of plucked hair may be rewarding, since there is an interval for most XBs during which cut hair may all be negative, but where the intradermal portion of the hair may harvest traces of the XB. Post-mortem, it is strongly recommended to take a hair sample prior to autopsy. The hair sample should be firmly tight together and tied with cotton, before being cut as close as possible to the scalp, making sure the scissors are leveled with the scalp. Still holding the sample tightly, the cut root ends of the sample must be aligned and carefully placed flat on a piece of aluminium foil, that is folded once or twice, with the cut root ends projecting ~ 15 mm beyond the end of the foil (Figure 15) (Flanagan et al. 2007). A
This procedure attempts to avoid misalignment of a hair sample necessary for segmental analysis (Flanagan et al. 2007). The sample should be stored in a tamper-proof envelope at room temperature until analyzed (Jenkins 2008). Putting hair into folded paper should be avoided, particularly in the presence of plucked hair, since the sticky hair roots will become fixed to the porous surface of the paper, and the strands will break at variable distances from the root (Karch 2008). The size of the sample to be collected is dependent on the purpose of the analysis. If a segmental analysis is desired, hair from a 1–2 cm area will typically yield ~ 50 mg of hair/ cm segments, which is the amount used for many GC-MS or LC-MS methods reported. Additional samples have to be collected if several analyses with different extraction techniques are desired. If the time for exposure is not an issue, smaller hair samples (100–200 μg) may suffice (Karch 2008). Deep freezing of hair samples have been shown to result in low XB concentrations, which is suggested to be due to XB diffusion from damaged keratin matrix during melting. If scalp hair is not available (or if it is excessively bleached or permed), alternative hair specimens may be collected from the pubic or axillary sites (Jenkins 2008). Hair contaminated with XB containing blood, vomit, or putrefaction fluid must not be taken, for it is well known that absorption of water or aqueous liquids are very rapid in enabling XBs to enter the hair. Alternatively, hair should be washed with water and then dried before sampling. It is wise to make a note that such contamination has occurred if it turns out that the blood contains high levels of XBs. Even sophisticated washing techniques are not capable of completely removing XBs from hair that is derived from these sources (Romano et al. 2001; 2003), and therefore specific metabolites of the XBs must be searched for. This is of 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 ~ 15 mm 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 et al. (2007).
398 R. J. Dinis-Oliveira et al.
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particular importance in the case of XBs that are likely to be in the environment because of the way they are consumed, such as cannabinoids and cocaine, where at least one metabolite should be detected (Cone et al. 1991; Uhl and Sachs 2004). This may present a problem, because normally the metabolites are more polar XBs, and have less affinity for hair matrix constituents (Cone et al. 1991; Uhl and Sachs 2004). Finger and toe whole nails or clippings In 1984, the presence of methamphetamine and amphetamine was demonstrated in nail clippings from habitual users, suggesting the usefulness of nails as forensic material (Suzuki et al. 1984). Since then, fingernails proved to be valuable specimens in suspected heavy metal poisoning, such as thallium, arsenic, or lead (Chen et al. 1999; Mandal et al. 2004; Brima et al. 2006; Lech 2006; Kile et al. 2007; Sanz et al. 2007). A number of other trace elements showed considerably more concentration in nails than in urine or blood (Karpas 2001; Karpas et al. 2005a; b; 2006). Other XBs detected in nails include cocaine and its major metabolites such as benzoylecgonine, ecgonine methyl ester, norcocaine and cocaethylene, 6-acetylmorphine, morphine, codeine, hydromorphone, oxycodone, hydrocodone, Δ9-tetrahydrocannabinol, and 11-nor-9-carboxy-Δ9tetrahydrocannabinol (Pichini et al. 1996; Engelhart et al. 1998; Palmeri et al. 2000; Engelhart and Jenkins 2002; de la Torre et al. 2004; Ragoucy-Sengler and Kintz 2005; Gray and Huestis 2007; Ali et al. 2008). Nails grow according to two different directions, length and thickness. Length growth rates of nails were reported to be 3–5 mm (Hamilton et al. 1955) and 1.1 mm (Bean 1953; 1980) per month for the fingers and toes, respectively. The thickening rate is constant and slow, with a mean value of 0.027 mm/ mm length (Johnson and Shuster 1993). Nevertheless, many different values are given in the literature by several authors and many factors can influence it. Growth is slowed with increasing age, cold climatic conditions, disease, and malnutrition, but it is faster in nail-biters. Even if there is considerable individual variation, the growth of the nail of the third digit seems to be greater than that of the others. No differences seem to occur between the rate of nail growth on the right and left hands or between men and women (Singh et al. 2005). It requires ~ 3–6 months for a whole nail to replace itself (Gupta et al. 2005). It is usually considered to be a third of average hair growth rate. Relatively little is known about the mechanisms of uptake and retention or XBs and metabolites in nail, making the interpretation difficult. Some literature data has shown that XBs are incorporated into nails by a double mechanism (Palmeri et al. 2000): a. Deposition into the root of the growing nail via the blood flow in the nail matrix; and b. Incorporation via the nail bed during growth from the lunula to the beginning of the free margin. Like hair, finger- and toenails accumulate XBs during longterm exposure, providing a retrospective window of detection,
even potentially longer than hair, and therefore this matrix has the potential to be a useful source for information about the XB exposure history of the decedent, especially when the scalp hair is too short or not available as a result of alopecia totalis or shaving of many body parts (Caplan and Goldberger 2001; de la Torre et al. 2004; Jenkins and Engelhart 2006; Lech 2006). Also in common with hair, care needs to be taken to ensure that external contamination is avoided or at least considered in any interpretation of results using these specimens. Unlike hair, nails do not contain melanin, and this may reduce XB incorporation. In addition, the variable and slower growth rate of nails, especially toe nails, as compared to hair, makes segmental analysis, and hence interpretation, hardly possible (Miller et al. 1997; Lemos et al. 2000; Maurer 2000; Palmeri et al. 2000; Caplan and Goldberger 2001; de la Torre et al. 2004; Jenkins and Engelhart 2006; Lech 2006; PayneJames et al. 2007; Mari et al. 2008). Ante-mortem, all finger- and toe-nails clippings should be collected and combined. Nail clippings from donors using Teflon-coated stainless steel scissors will be desirable to reduce contaminations. In post-mortem work, whole nails should be lifted from the fingers or toes. Some authors consider that toenails are better than fingernails because they are less exposed to external contamination. Whole nails or clippings can be stored at room temperature for very long periods, including years, without major degradation of incorporated XBs, enabling re-analysis. The stability of XBs in nails makes their analysis a good tool for post-mortem investigations, especially when it is impossible to perform other tests because of the lack of common body fluids or when decomposition of the remains can produce false results (e.g. negative tests due to the instability of analytes in body fluids or false positives from low specificity screening tests because of the presence of interferences) (Garside et al. 1998). Once XBs are incorporated into nails, levels remain relatively constant since fluctuations are not expected to occur due to changing of body metabolic activities, unlike the blood (Takagi et al. 1988). Nails that have been polished should not be sampled because elements added during the nail polishing may become an external element source (Sukumar 2006). In infants born from drug abusing mothers, nail analysis may offer some advantages over hair (Skopp and Potsch 1997). Compared to hair, that is not always present in the scalp of newborns or lost in significant amounts during the first months of life, and the analyst may encounter some resistance to collect it for cosmetic reasons, nails have the substantial advantage of always being present and considered ‘discardable’ material (Mari et al. 2008). Finally, the well-known drawback of nails analysis, that is external contamination by manipulation of XBs, is definitely unlikely in the case of a baby (Mari et al. 2008). On the other hand, in order to reflect fetal life, all the nail clippings of the first 3 months are needed, and these samples are not always gladly and continuously collected by the mother and, hence, not easily gathered (Mari et al. 2008). Nails are formed during the last trimester of pregnancy and are, thus, supposed to reflect the exposure only in this period. However, similarly
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to hair, incorporation of XBs in the nails of the fetus might be mediated by the fetal blood and/or by the amniotic fluid. Inasmuch as amniotic fluid is formed since the early stages of gestation, it is not impossible that the presence of XBs in nails is related to a XB exposure also during the first two trimesters (Mari et al. 2008). In a case of sudden infant death syndrome, cocaine was detected in nail clippings taken at autopsy from a 3-monthold infant, indicating a pre-natal XB exposure (Engelhart and Jenkins 2002). Owing to the vast consequences of this problem and to the importance of opportunely anticipating the onset of withdrawal symptoms in newborns, a number of bioanalytical procedures for monitoring drug abuse during gestation has been proposed, involving the collection of samples from either the mother (e.g. urine or hair) or the newborn (e.g. urine, meconium, hair, amniotic fluid) (Gray and Huestis 2007). Excised skin from injection sites Suspect injection sites could be excised and submitted for analysis, to support evidence of that route of administration. Skin, subcutaneous adipose tissue, and skeletal muscle should be sampled. The simple qualitative detection or even quantitative measurement of a XB in the excised tissue only evidences that the XB exposure occurred and not that it was necessarily injected. Sometimes it is forgotten that most XBs are distributed throughout the body from any route of administration, such that any piece of skin will contain some amount of the XB. For such measurements to be useful, a similar sample of the skin from another part of the body, not suspected to be an injection site, must be analyzed for comparison (Flanagan et al. 2007). Only if the concentration in the suspect site is substantially higher than that in the reference site, can significant conclusions be drawn (Karch 2008). Even then, a perfect injection may not cause persistent elevated XB concentrations at the intravenous injection site, in contrast to an intramuscular or subcutaneous site. Pleural effusion and pericardial fluid Several body fluids are available at autopsy, which may be used as an alternative specimen for XB detection, especially when other fluids are absent or are of reduced quality due to post-mortem processes. In addition, post-mortem blood XBs concentrations have been shown to vary depending on the sampling site. Therefore, although there are cases where blood is enough for analytic purposes, it may not be suitable for the resolution of the toxicological problems raised. In the laboratory, liquid specimens are easier to sample than tissue, and toxicology protocols designed for blood samples are easily adapted. Pleural effusion is a blood-stained fluid of the pleural cavity derived from liquefaction of surrounding tissues due to putrefaction. This putrefactive effusion forms in the pleural cavity and increases in volume as the blood in the vascular system disappears in the period of 1–2 weeks after death. It presents as a collection of dark, blood-stained and watery fluid. Thirty milliliters of the pleural effusion should be collected from the
pleural cavity and placed in a 30-mL-capacity plastic tube containing sufficient sodium fluoride to give a concentration of ~ 1% (Jones et al. 1999). Sims et al. (1999) showed that in 21 cases involving 20 different XBs, XB concentrations in pleural effusions were compared with those determined from blood specimens. Marked differences could be observed in cases involving ethanol, carbon monoxide poisoning, and flunitrazepam. In 70% of the cases, reasonable coincidence with pleural effusion/blood ratios in the range of 0.4–1.6 was found. XB levels in pleural fluid were also compared with concentrations in corresponding liver tissue. The authors suggested that bloodstained pleural fluid appears to be a suitable alternative material for estimation of XB levels in cases of advanced putrefaction, when no blood specimen is available, with partial or complete equilibrium of XB concentrations within the corpse (Sims et al. 1999). There is also little information on XB concentrations in pericardial fluid in spite of its sufficient amounts to be usable (Gibson and Segal 1978). Pericardial fluid is an ultrafiltrate of plasma with a very similar amount of proteins, which is contained within a tight compartment (pericardic sac) so that it is free of contamination by microorganisms. The usual volume currently taken at the time of the autopsy ranges from 5–20 mL. Moriya and Hashimoto (1999b) explored the usefulness of pericardial fluid as a specimen for toxicological analyses by comparing pericardial fluid with blood. The authors observed fairly good correlations between blood and pericardial fluid for all analyzed XBs and recommended that pericardial fluid should be added to the list of routine autopsy specimens. Pericardial fluid was sometimes better than blood when judging results, and it was suitable for quantitative estimations of XB concentrations, while cerebrospinal fluid, urine, and bile were considered useful for qualitative analysis. More recently, Contreras et al. (2006; 2007) showed the usefulness of analyzing drugs of abuse (morphine and cocaine) in pericardial fluid. Bone marrow There is a scarcity of research on the analysis for XBs in human bone marrow samples (Kojima et al. 1986; McIntyre et al. 2000; Raikos et al. 2001; Lafreniere and Watterson 2010). The first report occurred in 1978, when amitriptyline was identified in skeletonized human remains (Noguchi et al. 1978). Since then, most experiments have been undertaken using rabbit femoral bone marrow, which has shown linear relationships between bone marrow and peri-mortem blood XB concentrations for up to 24 hours for many XBs including tricyclic antidepressants, barbiturates, benzodiazepines, and ethanol (Winek and Jones 1980; Winek and Esposito 1981; Winek and Luhanik 1981; Winek et al. 1981; 1982; 1983; 1985; 1990; 1993; Winek and Janssen 1982; Winek and Susa 1982; McIntyre et al. 2000; Raikos et al. 2001; Schloegl et al. 2006b; Guillot et al. 2007; Kugelberg and Jones 2007). Bone marrow comprises vascular tissue present in the central cavities of bones (Jenkins 2008). There are two types of marrow: red and yellow, and these can differ in composition in different bones. Yellow marrow consists of a basis of
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400 R. J. Dinis-Oliveira et al. connective tissue supporting numerous blood vessels and inert adipose (fatty) cells (Jenkins 2008). The red marrow (or myeloid tissue) consists of a connective tissue (stroma) that supports clusters of hemapoietic cells, white blood cells, macrophages, and a rich vascular supply. Long bones receive a rich local blood supply, whereas short, flat, and irregular bones receive a limited supply through the periosteum. Normal human hemapoietic marrow is bright red in color and firm in consistency. There are some advantages to using bone marrow as an alternative tissue for analysis in Forensic Toxicology (Raikos et al. 2001). Bone marrow is often recoverable after skeletonization, it is encased in bone, and it has a high degree of vascularity and a lipid matrix that may act as a XB repository for lipophilic XBs. At this stage qualitative analysis using bone marrow seems to be very useful in documenting the presence of a XB that was not expected to be present. This acquires particular importance for XB detection in cases of advanced putrefaction, fragmentation, skeletonization, and exsanguinated human remains, and is useful when contamination of blood specimens is suspected in trauma cases. Nevertheless, although putrefaction is delayed in bone marrow, usually this specimen is not routinely considered as an alternative specimen in post-mortem toxicology, unless other specimens are unavailable. This can be explained mainly by the fact that, during putrefaction, bone marrow is transformed from spongy red marrow to a brown viscous liquid or paste-like substance compromising any interpretation from the quantitative point-of-view (Winek et al. 1983). Until now, no specifications exist concerning which bone should be used to obtain marrow. Bone marrow of ribs has been chosen (Schloegl et al. 2006b) due to being
easily accessible in routine autopsies, without changing the structure of the corpse in a relevant way, in contrast to bone marrow of vertebrae or long bones. Schloegl et al. (2006b) collected bone marrow tissue from the second and third rib on the right side. Routinely, in every autopsy the thorax is opened by cutting the ribs a few centimeters lateral of the sternum in their cartilaginous part and the sternum is taken out. For the collection of rib bone marrow, the rib has been cut ~ 5 cm from its distal end, which is approximately in the medioclavicular line where it is ossified (Figure 16) (Schloegl et al. 2006b). By compressing the ends of the remaining rib, the dark red bone marrow can be squeezed out of the bone. Bone Bone in humans has four shapes, namely long, short, flat, and irregular. Long bones have a central shaft, containing bone marrow, with expanded ends. Bones exhibit two types of tissue, solid dense compact bone and spongy bone, comprising a network of bony rods interspersed with spaces containing bone marrow. Both types of tissue are found in many bones, for example, in the humerus, compact bone forms the shaft and spongy bone is found in the ends or epiphyses (McGrath and Jenkins 2009). In cases of extensive decomposition, burnt, exsanguinations, or body fragmentation, only a few tissues may be available for examination. In these circumstances, bone (or teeth, which are one of the best preserved tissues) may be a useful specimen for potentially determining the role of XBs in medicolegal cases, but only as indicative of exposure. The reports that have been published thus far have consisted largely of 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 ~ 5 cm 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).
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Forensic toxicology 401 in the particular bone tissue sampled. There are no data suggesting that one anatomic region is better than another, but long bones such as the femur are easier to work with than smaller bones. The flat bone, iliac crest (Horak and Jenkins 2005), irregular bone, vertebrae, long bone, femur (McIntyre et al. 2000), and flat bones, sternum, and ribs (Benko 1985) have been selected. Bones should be cut into small segments (e.g. femur rings) or crushed (Drummer and Gerostamoulos 2002). Most XBs will be taken up by bone tissue and, therefore, unless volatile, will be detectable in skeletonized remains (Drummer and Gerostamoulos 2002). The degree of contact of XBs to the bone structures depends on the anatomical location of the bone and the local blood supply. Long bones receive higher blood supply comparatively to the short, flat, or irregular bones, which receive more superficial supply through the periosteum. This intrinsic property advocates the use of long bones (i.e. femur) instead of others. For certain XBs the interpretation of concentrations is relatively easy since either the normal or reference values are well established (e.g. heavy metals), or the XB should not be present in any concentration (e.g. strychnine), while, for others, such as pharmaceutical drugs or drugs of abuse, the interpretation is problematic because of limited reference levels (McGrath and Jenkins 2009). In addition, it should be recognized that bone is continuously remodeled. Therefore, XBs incorporated in bone tissue over time will be liberated and re-delivered to the blood, which means that a negative detection in bone does not rule out an exposure, and a positive detection will not give very much information about time for exposure. Indeed, XBs distributed to bone will almost certainly remain here longer than in blood due to the relatively slow turnover of the bone, increasing therefore the time frame for detection. Comprehensive data on post-mortem XB findings in bone were provided by McIntyre et al. (2000). Tricyclic antidepressants, as well as non-tricyclic drugs such as mianserin and moclobemide, antipsychotics (chlorpromazine, thioridazine, and clozapine), benzodiazepines (diazepam, oxazepam, and temazepam), and major corresponding metabolites, were identified in sections from human femoral bone and bone marrow. The majority of targeted XBs generally matched qualitatively to the blood results. The concentrations of primary metabolites were lower compared to those of the parent XB. In a rabbit model, methamphetamine was always detectable in bones that were kept under various conditions over a 2-year period (Nagata et al. 1990). Elevated aluminum levels in liver, brain, and bone specimens helped to diagnose the most probable cause of death in dialyzed patients (de Wolff et al. 2002; Cengiz et al. 2006). Some important conclusions were recently ascertained by McGrath and Jenkins (2009). The authors observed that several factors influence the deposition of XBs in this matrix, including acute vs chronic exposure, distribution at the time of death, site of bone collection and bone type, and physicochemical characteristics of the XB. XBs with short half-lives or those inherently unstable, such as 6-acetylmorphine and olanzapine, may not be stored in bone. In addition, polar metabolites and highly protein-bound
compounds may be transferred from blood to bone to a lesser extent. Some XBs, maybe reflecting exposure chronicity, were detected in bone, but not the corresponding blood. Similar to previous findings obtained for some XBs (McIntyre et al. 2000) there appeared to be no relationship between a blood XB concentration and the likelihood of a positive finding in bone, and high blood concentrations did not always result in elevated concentrations in bone. In a pilot study, morphine and codeine were detected in human teeth from individuals that had died of heroin overdose (Cattaneo et al. 2003). However, as for other bones, the rate and degree of entry into the various structures of teeth (dentine and enamel) is not known and likely to be slower than for long bones due to the lesser degree of vascularization. Further studies are required to determine the dose, blood concentration, and frequency of XB use necessary for deposition in bone (McGrath and Jenkins 2009). Controlled studies investigating drug disposition in skeletal tissues are more easily done using animal tissues as opposed to human autopsy tissues. However, the direct applicability of such studies to the interpretation of forensic analyses is still limited. Entomological specimens (entomotoxicology) When decomposition prevents traditional specimens, such as blood, urine, or solid tissues, from being obtained, homogenized fly larvae (maggots), usually of Calliphoridae (blowfly), Sarcophagidae (flesh flies), and Muscidae (house flies) families have proved to be useful alternative specimens in which XBs may be identified (Byrd and Castner 2001). They are highly motile, strong-flying insects, and are typically the first to reach the dead body, often within minutes of death (Byrd and Castner 2001). Depending on temperature, larvae may be present as soon as 1 to 2 days after death (Byrd and Butler 1996; 1997; 1998). XB concentrations in larvae have been found to depend on the tissue the larvae had fed on as well as on their stage of development, which is influenced by the type of XB present in the corpse (Kintz et al. 1990b; Wilson et al. 1993). Indeed, various studies point to the influence of XBs in the development of larvae, which can compromise an accurate estimation (either by excess or deficit) of the postmortem interval (Goff et al. 1991; Arnaldos et al. 2005; George et al. 2009). It appears to be critical that larvae collected for XB analysis from a decomposed body are frozen and analyzed as soon as possible after collection, since larvae rapidly eliminate XBs when removed from a food source (Karch 2008). Nevertheless, even under refrigerated conditions, when larvae are in a state of diapause, slow bioelimination of XBs still occurs over the course of several weeks (Sadler et al. 1995). The choice of where larvae are best collected from the body needs further study (Karch 2008). Interpretation of positive findings seem to be most useful if the larvae are collected at the site of their food source, such as any remaining muscle or liver, under the premise that XBs detected in fly larvae feeding on a body can only have originated from the tissues of that body (Pounder 1991; Pounder et al. 1996a). This assumption seems to be supported by earlier studies where a quantitative relationship
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402 R. J. Dinis-Oliveira et al. was suggested between the morphine concentrations in the larvae and the livers on which they fed (Introna et al. 1990; 2001). By contrast, other studies suggest that the analysis of fly larvae provides only qualitative data (Kintz et al. 1990a; b; c). However, in these studies, larvae specimens were obtained without taking into account that larvae XB concentrations are based on the tissue on which they fed (Pounder 1991; Pounder et al. 1996a), since specimens were collected from multiple sites and they were pooled before analysis. Nevertheless, at this stage, due to the wide variations demonstrated in the various studies cited above, it seems more reasonable to consider that accumulation of XBs in larvae is unpredictable and quantification unreliable (Sadler et al. 1995). Even if used only for qualitative analysis, larvae toxicological analysis could play an important part in detecting XBs and can contribute to establishing the cause, the manner, and the mechanism of death. To avoid environmental (surface) contamination, a source of interpretive error, larvae should be washed with deionized water prior to analysis (Gagliano-Candela and Aventaggiato 2001). For analysis, larvae are homogenized and processed in the same manner as human tissues. XBs extraction techniques from chitinized insects are similar to those performed on hair samples. The first reported use of fly larvae in XBs analysis occurred as recently as 1980 and involved a phenobarbital case (Beyer et al. 1980). Since then numerous XBs have been identified in fly larvae including benzodiazepines, tricyclic antidepressants, opiates, cocaine, and the organophosphate, malathion (Gunatilake and Goff 1989; Kintz et al. 1990a; b; c; 1994; Nolte et al. 1992). Gagliano-Candela and Aventaggiato (2001) published an interesting review on the XBs detected in entomological specimens and on experimental studies.
Reporting results and interpretation aspects One of the major concerns that must be always present is that the results of a toxicological investigation may be included in court testimony. In addition, as an expert in the field of intoxications, the forensic toxicologist may be asked to give an opinion either by a report or in the courtroom. Often, the defense will call its own experts to dispute the opinions given by the plaintiff or prosecutor’s toxicologist. Cases involving XBs, for several times become a debate of experts and such proceedings can be quite intense, with the jury left to decide which expert opinion seems most likely to be true. In assessing the evidence of the analytical toxicological results, the courts are concerned especially with the experience of the analyst, the ability to prove continuous and proper chain of custody compliance, and the analytical methods used. The following are some examples of the questions or comments that the forensic toxicologist might be asked by medical, legal, or law enforcement professionals (Skopp 2004; Stripp 2006): a. Was the driver intoxicated at the time of the accident? b. Was XB-induced psychosis a likely explanation for a person’s bizarre and violent behavior?
c. What was the contribution of alcohol or other XBs for a person to fall down a flight of stairs? d. In the case of a death in a fire, was the victim burned? Is the death consistent with smoke inhalation? Was the victim alive or already dead when the fire occurred (Dinis-Oliveira et al. 2010)? e. Were drugs of abuse used to incapacitate a girl during an alleged date rape? f. Was a XB used to commit suicide? g. Was a person murdered with a XB? h. Is the concentration of the XB able to explain the toxic symptoms or the fatality? i. What was the route of XB exposure? j. To comment on acute or chronic intake/exposure; k. To classify the concentrations as therapeutic or toxic; or l. To roughly estimate the time interval between exposure/ intake and death. The forensic toxicologist must be able to provide accurate and concise answers to these queries using language that is easily understood by lawyers, jurors, and judges. Furthermore, the opinions expressed by the toxicologist must always be impartial and based only on the scientific facts involved. Comprehensive information already prior to sampling is an important precondition that even allows answering such complex questions and supporting reliable interpretation of the analytical results obtained from ante-mortem or postmortem material. A standard report format is very helpful and results should be reported in appropriate units, preferably written out in full to avoid confusion (e.g. between mU/L and μU/L), indicating the measurement uncertainty if desirable, with appropriate reference data or information to assist the requester in understanding their significance (Jickells and Negrusz 2008). This may, for example, be a reference to legislation, production criteria, and published papers in the scientific literature, or the expected values for clinical investigations (Jickells and Negrusz 2008). Special care is required when comparing results, especially when it is needed to choose the correct relative molecular mass (Mr, ‘molecular weight’) if the XB is supplied as a salt, hydrate, etc., since this can cause great discrepancies, especially if the contribution of the accompanying anion or cation is high (Flanagan and Connally 2005). Analytical measurements should be reported in terms of the free acid or base and not the salt. Care is also needed in comparing blood (e.g. mg/L) and tissue (e.g. mg/ Kg) concentrations: 1 L of water has a mass of 1 Kg at 4ºC, but slightly variations occur at different temperatures. The debate over choice of units has been reviewed by Flanagan (1998; 2004). An example of a Forensic Toxicology report is given in Figure 17. It is important to have always the conscience that there are no ‘absolute’ rules for the interpretation of toxicology results since it is not a simple matter, as becomes obvious
Forensic toxicology 403 by the revisions of literature performed by several authors (Levine et al. 1990; Flanagan 1998; 2004; Drummer and Gerostamoulos 2002; Drummer 2004; 2007a; Flanagan and Connally 2005; Kugelberg and Jones 2007). The more information that is available to, and considered by, the interpreter, the more likely are the conclusions reached to be accurate. In the courtroom, lawyers, jurors, and judges often view all science, including the forensic sub-specialties, in absolute terms. Certainly, if the toxicologist does his or her job properly, the laboratory findings will have the required accuracy (Karch 2008). It is also obvious that the interpretation of any toxicological results will be no more reliable than the analytical result itself. All interpreters must know the limitations of the testing and the following questions should be taking into account (Karch 2008):
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a. Was the standard material used to prepare the calibrators pure and correctly identified? b. Was the salt or water of crystallization properly taken into account? c. Was the calibration properly prepared and validated in the range where the specimens were measured? d. Was the assay adequately verified by quality control samples? e. Was the assay sufficiently specific? f. Could other XBs or metabolites have interfered with analysis of the specimen, either by obscuring the target analytes or by increasing the apparent concentration? g. If the specimen was analyzed only once, what was the potential for accidental contamination? h. Was there a matrix effect? i. Was the recovery of the XB from the specimen the same, relatively, as from the calibrators? j. Even using similar matrix calibrators (e.g. whole blood) is not necessarily a guarantee of the correction of matrix effects when using post-mortem blood, since, by its nature, it is variable from case to case, or even from site
to site within the same cadaver. The extraction efficiency of the XB, metabolite or internal standard from outdated blood bank blood may sometimes be markedly different from decomposed case blood. Although it is practically impossible to know the ‘absolute’ or true concentration of XB in a specimen, the degree of confidence increases with the specificity of the analysis, with replication, or in some cases by applying multiple analytical methods of different physical or chemical principles. The use of GC-MS with multiple ion monitoring and stable isotope (e.g. deuterated) labeled internal standards will usually provide a higher degree of confidence in the accuracy of the analytical result than using an immunoassay procedure (Karch 2008). The completeness of the analysis should also be considered. It is impossible to test for every single XB during routine screening tests. However, a careful review of the medications or other potential XB available to the victim should assist the laboratory in focusing the toxicological analysis. Attempts to interpret toxicology findings solely on the basis of so-called normal or reference ranges are irresponsible. More erroneous becomes the interpretation in the field of post-mortem Forensic Toxicology. Probably, there is no forensic toxicologist or pathologist alive who has not used published tables as a reference when trying to interpret postmortem blood concentrations. Tables of such values became a necessary evil due to the sheer volume of medical and forensic literature. However, they unfortunately perpetuate the myth that post-mortem toxicology results can be interpreted solely using, or heavily relying on, so-called ‘therapeutic’, ‘toxic’, and ‘fatal’ ranges (Karch 2008). Although tables of XB concentrations can serve as a useful reference point, it should be borne in mind that many of the values in these tables are derived mostly from serum or plasma data from living patients (Schulz and Schmoldt 1994; 1997; 2003; Winek et al. 2001), that the ranges are seldom referenced to published cases, and that they are not take into account or state other variables such as post-mortem redistribution (also referred to as the ‘toxicological nightmare’ (Pounder and Jones 1990)), diffusion and metabolism, tolerance, time of survival after
TOXICOLOGICAL REPORT Nº2010/_____ Victim name:________________________________________________________________ ________________________________________________________________ Requested by: Toxicological parameter
Analytical Sample (refer the Resultand units technique and anatomic place of limit of detection blood collection) (LOD)
Forensic analyst signature
Commentary:_______________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ Date, place and signature of the service director:__________________________________ _________________________________________________________________________ Figure 17. An example of a Forensic Toxicology report.
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404 R. J. Dinis-Oliveira et al. intoxication, or the presence of other XB (interactions), natural disease, or injury. In addition, specific references to case data and further information are lacking in most instances. For example, interpretation of opioids concentrations may be very dependent on how long the person has been exposed to the XB and at what dosage. The inappropriate use of tables can result in over- and under-estimation of the potential toxicity of a XB depending on the degree of tolerance developed, natural disease, route of administration, and whether other XBs are present. In these cases setting ‘lethal’ post-mortem levels is very difficult, since the range of post-mortem overdose levels often overlap with those seen in patients on chronic opioid treatment. Post-mortem concentrations have been over-interpreted in the past, and good evidence should be required before ‘lethal concentrations’ are defined in the future. The same rationale is obvious when analyzing literature data concerning ethanol. The lethal concentration of ethanol has sometimes been reported as ‘above 500 milligrams per 100 milliliters’ but a patient was still talking when her serum ethanol concentration was > 1500 mg per 100 ml, three times this ‘fatal’ value (Johnson et al. 1982). Therefore, while these tables may be of some (and not all) use in clinical toxicology, they are of very limited value for the interpretation of post-mortem toxicology results and can be very misleading. Nevertheless, some important compilations providing reference levels on post-mortem femoral blood samples have been published (Druid and Holmgren 1997; 1998; Reis et al. 2007). Such data are important and additional compilations using this approach are obviously encouraged. Kinetics is an invaluable tool to help understand the time course of XB in the body. In the living, it can be used to determine duration of action, inter-individual differences in peak plasma concentrations and clearance, and the likely effectiveness of different pharmaceutical formulations. However, rarely can kinetics be applied successfully to post-mortem toxicology. The kinetics of all XBs in the body is characterized by absorption, distribution, metabolism, and excretion. All these steps affect the concentrations of XBs and therefore the interpretation of ante-mortem and post-mortem analytical toxicology results. Nevertheless other issues must be considered when interpreting post-mortem toxicological results, since the human body is not a static entity after death. Post-mortem redistribution represents a special concern that misled the interpretation in post-mortem Forensic Toxicology. Toxicologists participating in forensic cases involving XBs likely to undergo post-mortem redistribution must be aware of its potential contribution to the post-mortem XB concentration. In addition, often no ante-mortem or peri-mortem XB blood is available for analysis, and clinical information about the deceased at the time of death may not exist if no medical attention was provided. Post-mortem redistribution reflects the diffusion and redistribution of XBs occurring in the body in the interval between death and autopsy sampling (Cook et al. 2000) along a concentration gradient. Redistribution generally refers to the release of XBs from areas of higher concentration (e.g. from binding sites in cells or tissues) and subsequent passive diffusion into interstitial
fluid, through the capillaries and into the larger blood vessels of those organs, resulting in higher concentrations of the XBs in surrounding tissues than true concentrations at the time of death. Once cells die, their membrane integrity is lost, and the cells leak their contents (XBs, electrolytes, etc.) into the extracellular space. In these cases, post-mortem levels in central (cardiac) blood can be several times higher than they were ante-mortem, due to passive diffusion from myocardial cells into blood in the cardiac chambers or diffusion from adjacent lung tissue into the great vessels in the thorax (aorta, inferior vena cava, etc.) (Yarema and Becker 2005; Holland 2009). Post-mortem diffusion generally refers to the diffusion of XBs along a concentration gradient, from an area of high concentration to an area of low concentration located nearby. The usual scenario is when a high concentration of XB in the stomach contents (e.g. after an overdose) causes elevated concentrations of the XB in nearby tissue (e.g. proximal lobe of the liver) or blood. For example, in life, ethanol is absorbed from the small intestine rather from the stomach. However, after death the stomach wall becomes permeable to ethanol, which then diffuses into adjacent tissue and blood vessels (Pounder and Smith 1995; Pounder et al. 1996b). Such diffusion through previously impermeable barriers may be most important for small non-polar molecules (Pelissier-Alicot et al. 2003). Much is still unknown about the extent to which post-mortem changes in XB concentration occur, and the different XBs affected. The likelihood that post-mortem samples of blood reflect ante-mortem XB levels will depend on the circumstances of the death and post-mortem sampling, as well as whether post-mortem redistribution is likely to take place. The characteristics of the XB itself, which will influence the likelihood of post-mortem redistribution, are the volume of distribution, the pKa, and the lipophilicity (Pounder and Jones 1990; Pelissier-Alicot et al. 2003; Yarema and Becker 2005; Holland 2009). If the volume of distribution of the XB is high, ie a volume of distribution > 1 L/Kg, then that XB is more distributed to tissues, and post-mortem redistribution is more likely to occur. The pKa of the XB is defined as the pH at which the XB is 50% ionized. A basic XB, i.e. pKa > 7, will exhibit more post-mortem redistribution. This can be explained by two mechanisms: (i) basic XBs are more prone to concentrate in tissues before death, and (ii) the contents of a cell are largely aqueous and become acidic after death. Since a basic XB will be progressively more ionized in an increasingly acidic medium, after cell lysis occurs, basic XBs will distribute more readily as a result of being transported in the acidic fluid in which they are dissolved. Highly lipophilic XBs are also more distributed in tissues, and are therefore more likely to exhibit post-mortem redistribution. A comprehensive discussion of the possible mechanisms for post-mortem redistribution and the XBs affected was published by Pelissier-Alicot et al. (2003) and Yarema and Becker (2005). Another topic that must be considered when interpreting XB concentrations is the sum, or even more complex, the synergy of the effects of all of the XB detected. In the majority of cases in clinical and Forensic Toxicology, more than one XB is involved. Multidrug therapy and abuse is
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Forensic toxicology 405 prevalent, and this, together with the added problems of self-medication with over-the-counter drugs and the widespread use of alcohol, makes interpretation of data even more complicated. Several deaths often involve multiple drugs of the same type (e.g. benzodiazepines or narcotics), individually present in ‘therapeutic’ amounts, and often in combination with alcohol. Interpretation of blood XB concentrations in these cases has to take into account any disease that may be present, and the total amounts of XBs and alcohol. XB interactions can be divided into two types: pharmacokinetics and pharmacodynamics. Both divisions can originate addition, synergism, potentiation, and antagonism interactions. Artifacts of absorption and distribution must also be recognized when interpreting post-mortem blood concentrations. For example, it is quite common to find grossly elevated concentrations of lidocaine in cases where resuscitation has been unsuccessfully attempted (Moriya and Hashimoto 1997c). Forensic toxicologists should be always aware of such a phenomenon. Concentrations may be 2–5times those normally considered therapeutic when lidocaine 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 established, very high local concentrations will result in the cardiac blood. These could be interpreted as ‘fatal’ unless all the circumstances are considered. In addition, jelly lidocaine formulations are usually used at endotracheal intubation in emergency medicine, being frequently detected in blood of cadavers that had received cardiopulmonary resuscitation (Moriya and Hashimoto 1997c; 1998a; Pounder 1997). Devices that automatically deliver medication by the parenteral route (such as transdermal patches) can lead to artificially extremely high post-mortem local blood concentrations (misinterpreted as an overdose) unless they are switched off or disconnected quickly (Anderson and Muto 2000; Solarino et al. 2010). Since these patches rely primarily on passive diffusion across a rate-limiting membrane for XB delivery, the concentration of the analyte in the local area will continue to rise after death, albeit at a slower rate. Since blood circulation through the skin obviously stops after death, the XB will no longer be transported away, except by diffusion, allowing a local build-up of XB (Jickells and Negrusz 2008; Karch 2008). Despite these concerns, some aspects of interpretation remain relatively straightforward. A negative result is not sufficient to establish that the XB is not implicated in the intoxication case. Negative results are sometimes assumed because standard screening tests have shown no suspicious results or the XB disappears before analysis. Additionally, a negative result below a defined limit of detection can be interpreted as lack of acute exposure to that analyte or non-compliance in the case of therapeutic drugs (Karch 2008). A positive qualitative analysis never solely provides conclusions of the involvement of an XB in the intoxication, but is only one of many pieces of evidence. For example, minute quantities of cyanide, arsenic, lead,
and dichlorodiphenyltrichloroethane (DDT) are regularly ingested from food sources or inhaled as environmental contaminants and retained by the human body, but in amounts that are insufficient to cause obvious deleterious effects (Klaassen 2008). Ethanol represents another typical example, since it is produced after death (Kugelberg and Jones 2007). Conversely, blood XB concentrations that exceed therapeutic concentrations by 10–20-times are consistent with intoxication or death, excluding an obvious contamination problem. In addition, the higher the parent XB-to-metabolite ratio, the more likely an acute intoxication has occurred (Karch 2008). Finally the possibility of endobiotics involvement in the intoxication should not be disregarded (Lam et al. 2006; Chambellan-Tison et al. 2007). A compound apparently innocuous as pure water will, if ingested in sufficient quantity (as occurs in schizophrenic patients suffering from polydipsia), can cause incapacitating electrolyte imbalance, hyponatremia, confusion, rhabdomyolysis, systemic seizure, and edema or even death (Vieweg et al. 1985; Chen and Huang 1995; Arieff and Kronlund 1999; Hayashi et al. 2005).
Concluding remarks One of the main functions of the forensic toxicologist is to produce results and to interpret them in order to be used in criminal prosecutions, which implies rigor to survive court cross-examination and medicolegal scrutiny (Drummer 2007b). As a general view, probably extended to all branches of the forensic sciences, much of the information transmitted in Forensic Toxicology has been obtained from case studies described at meetings or in published works (Drummer 2007a). Interpretation has therefore been empirical, with typical intoxication data found in the literature serving as benchmarks (Fanton et al. 2009). Collectively this has formed much of the basis of knowledge in this discipline, which led to criticisms from some groups, of other medical disciplines in medical faculties, regarding the lack of clear evidence of some conclusions that are drawn in specific cases and the bias that can lead to poor quality and quantity of research (Drummer 2004; Guzelian et al. 2005). Indeed, Forensic Toxicology has different aims and relevant intersections with arts and socio-scientific disciplines (such as law) in comparison with Clinical Toxicology, a fact that increases the number of variables to be considered (Madea et al. 2007). In addition, controlled randomized, prospective cross-sectional, cohort, or case-control studies can only rarely be applied in Forensic Toxicology. Specifying the optimum size of a sample in order to attribute credibility to the results is also always problematic in Forensic Toxicology. For many laboratories, sample sizes of fewer than 10 are common, but when investigations of cases are conducted with the view to compare them and illustrate common features, sample sizes will need to be much higher. For example, understanding the association between illicit drug use and crash risk has required samples sizes of many hundreds to thousands (Mura et al. 2003; 2006). In the literature, there
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406 R. J. Dinis-Oliveira et al. are many studies that have provided significant knowledge regarding the specimens and amount to be collected. Some of these examples were discussed and harmonized guidance is attentively given in this paper. It is recommended that medical examiners/pathologists, and if necessary the police investigators or judicial authority, discuss the case with toxicologists in advance of collection to ensure that the most appropriate specimens are collected. Since resampling is rarely possible, it is recommended to collect a minimum set of specimens. As referred for several times, ante-mortem serum or plasma and post-mortem peripheral whole blood (obtained from the femoral vein) are the forensic toxicologist’s most useful specimens for toxicological analysis. Nevertheless, it is obvious that conducting toxicological analysis will vary from case to case (Klaassen 2008) and will depend on the policy of the jurisdiction. The application of robust and properly validated analytical methods will provide the basis for the most correct interpretation of toxicology findings. As discussed above, results must be interpreted with regard to all of the available information, including medical history, ante-mortem or peri-mortem toxicological results, information from the scene, autopsy findings, nature, and exact location of the post-mortem samples collected, and the circumstances of the death. Some of the fundamental differences of investigations carried out on living, in contrast to dead, were assessed throughout this paper. In this final remark, it is important to remember that investigations on post-mortem specimens creates a number of additional challenges to the forensic analyst that are not observed in ante-mortem toxicology, namely problems related to autolysis, post-mortem redistribution, decomposition, or absence. Therefore, the specimens are often less than ideal, which means that post-mortem results are only reliably interpreted after weighing all of these variables. Even then, it must be admitted that adequate interpretation of some results is simply not possible based on the available information (Karch 2008). In many respects, the desirable underlying approach to the interpretation of post-mortem XB concentrations is not much different from that used a century ago: a good scene investigation, medical and laboratory toxicological investigation, based on the history and circumstances surrounding the death, and the application of common sense. Accordingly, there is still much to learn, particularly in what concerns to the individual contributions of multiple XBs, the contribution of natural disease in XB-positive cases, and the mechanisms responsible for the post-mortem changes in XB concentration.
Acknowledgements The authors are also thankful to the Forensic Toxicologists and Pathologists of the Departments of Forensic Toxicology and Pathology of the Portuguese North Branch, National Institute of Legal Medicine, I.P., for the always present helpful collaboration and continuous discussions. A special acknowledgement to Dr Dina Almeida for fruitful help in performing autopsy photographs.
Declaration of interest Ricardo Dinis-Oliveira acknowledges FCT for his Post-Doc grant (SFRH/BPD/36865/2007). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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