**2. Poisoning**

In the field of forensic toxicology, concepts of poisoning and adverse reaction are encompassed in the concept of intoxication according to a unitary criteriological vision; always oriented at resolving a fundamental query concerning the nexus between the action of one or more xenobiotics and the functional and/or morphological harm on the organism until its death. Substances considered *poisons* are those capable of causing damage even in low doses. Even drugs, and in any case all xenobiotics, can exert a poisonous action for an absolute overdose, for pharmacokinetic-pharmacodynamic synergies or for endogenous or exogenous factors.

The classification of poisons in Forensic Toxicology is traditionally dependent on the basis of chemico-physical characteristics of substances and on the consequent possibility of extractions from biological fluids with a method specific for classes. Therefore they have six groups: 1) *poisons in a gaseous or vaporous state* that when inhaled cause intoxication (carbon monoxide, hydrogen sulphide, ethylic ether, chloroform, etc.); 2) *poisons in a liquid state prone to volatility*

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(benzene and derivatives, glycols, aldehydes, essential oils of some plants, etc); 3) *acids and strong bases* (hydrochloric, sulphuric and nitric acids, sodium or potassium hydrate, etc.); 4) *inorganic anions* (permanganates, chromates); 5) *metals* o salts of metals (arsenic, thallium, mercury, lead); 6) non volatile *organic compounds* of acidic, neutral or basic nature (the most of drugs of synthesis, alkaloids, narcotics, insecticides, agrochemicals etc.). It must be considered, however, that some toxins and many drugs, with a polypeptidic (for example insulin) or protein structure (for example erythropoietin), have physical and analytical chemical pecu‐ liarities that do render their extraction and analysis and also their classification more complex.

In many cases, poisoning represents an unknown event that can be demonstrated only through a balanced criteriology for obtaining a differential diagnoses, often inopportunely overlooked in the number of tests to be executed in cases of unexpected death. All of this leads to an underestimation of poisoning incidence in the field of so-called *medico-legal pathologies*. Included in this field are *drug related deaths (DRD)*, a widely investigated and delineated phenomenon in terms of incidence, prevalence and social impact. The multiplicity of patho‐ logical factors that contribute to its cause is demonstrated by a complex definition elaborated by a German study group [1], according to which DRD term groups deaths due to *"accidental or intentional overdoses, long term drug abuse, suicide associated to toxic dependency, lethal accidents influenced by the use of drugs".* DRD is a pathological epiphenomenon reductively denominated an "overdose" or "adverse reaction", originating from variable physiopathological pharma‐ cotoxicological and immunological mechanisms, whose genesis has not found and cannot find a comprehensive explanation in a morphological or chemical-toxicological cause, considered in isolation.

The phenomenon also assumes particular connotations in relation to the quali-quantitative heterogeneity of active drugs, the peculiarity of pre-existent psychophysical conditions, the unpredictability of pharmacotoxicological interactions from poly-drug abuse, capable of inducing a sometimes exponential increase of the risk of lethal *adverse reactions*.

Post-mortem diagnosis of acute and/or chronic intoxication is a paradigmatic example of the necessity to correlate circumstantial, clinical (documentary, anamnestic, objective), necroscop‐ ic (autoptic, histologic, immunohistochemical, microbiological, thanatochemical) and chemi‐ cal toxicological data, obtained following an accurate methodological approach; the gathered data must successively be the object of an evaluative forensic toxicological criteriology. In this view the existence of standardised methodological protocols finds its reason, applied to the resolution of cases where an acute or chronic intoxication of forensic concern is suspected. The necessity of uniform autoptic procedures has been expressed in Recommendation N. 99 of the Committee of Ministers of the Council of Europe, relative to the harmonisation of the rules concerning the legal medical autopsy [2].

The circumstantial data draw on the spatio-temporal circumstances of the presumed "contact" with the toxicant; they can derive from police investigations, testimonial depositions, technical inspections, site inspection reports, etc., and are for example the insurgence of pathological phenomenon or of death following the ingestion of foods or beverages, or contact with chemical substances, or the retrieval of pharmaceutical provisions or paraphernalia in the location where a corpse is discovered.

The clinical data refer to sanitary documentation that is examined to evidence any general or local disorder, in order to outline the objective symptoms characterizing an intoxication. In this view an appropriate toxicological evaluation needs a profound knowledge of the spectrum of biological effects of a specific chemical substance, of the intensity of individual effects dependent on the dose (*dose-effect relationship*) and on the frequency or probability of the appearance of a dose-dependent effect in a determined population (*dose-response relationship*).

(benzene and derivatives, glycols, aldehydes, essential oils of some plants, etc); 3) *acids and strong bases* (hydrochloric, sulphuric and nitric acids, sodium or potassium hydrate, etc.); 4) *inorganic anions* (permanganates, chromates); 5) *metals* o salts of metals (arsenic, thallium, mercury, lead); 6) non volatile *organic compounds* of acidic, neutral or basic nature (the most of drugs of synthesis, alkaloids, narcotics, insecticides, agrochemicals etc.). It must be considered, however, that some toxins and many drugs, with a polypeptidic (for example insulin) or protein structure (for example erythropoietin), have physical and analytical chemical pecu‐ liarities that do render their extraction and analysis and also their classification more complex. In many cases, poisoning represents an unknown event that can be demonstrated only through a balanced criteriology for obtaining a differential diagnoses, often inopportunely overlooked in the number of tests to be executed in cases of unexpected death. All of this leads to an underestimation of poisoning incidence in the field of so-called *medico-legal pathologies*. Included in this field are *drug related deaths (DRD)*, a widely investigated and delineated phenomenon in terms of incidence, prevalence and social impact. The multiplicity of patho‐ logical factors that contribute to its cause is demonstrated by a complex definition elaborated by a German study group [1], according to which DRD term groups deaths due to *"accidental or intentional overdoses, long term drug abuse, suicide associated to toxic dependency, lethal accidents influenced by the use of drugs".* DRD is a pathological epiphenomenon reductively denominated an "overdose" or "adverse reaction", originating from variable physiopathological pharma‐ cotoxicological and immunological mechanisms, whose genesis has not found and cannot find a comprehensive explanation in a morphological or chemical-toxicological cause, considered

The phenomenon also assumes particular connotations in relation to the quali-quantitative heterogeneity of active drugs, the peculiarity of pre-existent psychophysical conditions, the unpredictability of pharmacotoxicological interactions from poly-drug abuse, capable of

Post-mortem diagnosis of acute and/or chronic intoxication is a paradigmatic example of the necessity to correlate circumstantial, clinical (documentary, anamnestic, objective), necroscop‐ ic (autoptic, histologic, immunohistochemical, microbiological, thanatochemical) and chemi‐ cal toxicological data, obtained following an accurate methodological approach; the gathered data must successively be the object of an evaluative forensic toxicological criteriology. In this view the existence of standardised methodological protocols finds its reason, applied to the resolution of cases where an acute or chronic intoxication of forensic concern is suspected. The necessity of uniform autoptic procedures has been expressed in Recommendation N. 99 of the Committee of Ministers of the Council of Europe, relative to the harmonisation of the rules

The circumstantial data draw on the spatio-temporal circumstances of the presumed "contact" with the toxicant; they can derive from police investigations, testimonial depositions, technical inspections, site inspection reports, etc., and are for example the insurgence of pathological phenomenon or of death following the ingestion of foods or beverages, or contact with chemical substances, or the retrieval of pharmaceutical provisions or paraphernalia in the

inducing a sometimes exponential increase of the risk of lethal *adverse reactions*.

in isolation.

86 Toxicology Studies - Cells, Drugs and Environment

concerning the legal medical autopsy [2].

location where a corpse is discovered.

The anatomopathological data are intended to reveal morphological modifications, patho‐ gnomonic and not, of the organs and systems of the deceased for suspected (acute or chronic) intoxication. In this field the medico-legal literature is lacking thorough studies, extended to all the organs and to the diverse modes of poisoning. In some cases, a macro and microscopic observation can enable identification of toxic-related harmfulness, also non-specific, apt to explain the death or direct the chemical-toxicological investigation. The examinations must include observance of cadaveric phenomena to capture changes in the normal coloration of hypostatic stains, detection of changes in rigidity and evolution of putrefactive phenomena, external examination (presence of jaundice, alterations in cutaneous appendages) and autopsy. A complete evaluation of macroscopic investigations, integrated by the optic and electronic microscope analyses, as well as by specific immunohistochemical techniques, may enable comprehension of the ethiopathogenesis and physiopathogenesis of the intoxication. In particular, it is important to examine the whole encephalon and the heart [3], as well as anatomical organs and structures sometimes undervalued, such as the carotid body, bone tissue and sexual organs. Examination of the *carotid body* provides indications as to possible alterations resulting from chronic hypoxic states and abuse of opiates [4]; *bone tissue*, as to alterations of bone marrow due to toxic or neoplastic diseases; *sexual organs*, as to testicular atrophy attributable to chronic exposure to steroid hormones; the *lymphonodal stations* to distinguish hypertrophy of an infective, inflammatory or neoplastic origin.

Theanatomo-pathologicalfindingsallow,therefore,toaddress investigations intwodirections:


The chemical-toxicological data are needed for the qualitative and quantitative demonstration of the presence of the toxicant in the biological fluids and in the tissues collected during the clinical or necroscopic ascertainment.

Retrieval of toxic substances in the living or dead body constitutes, in general, the most important criterion, often decisive to the diagnosis.

The chemical-toxicological analysis should be articulated according to two directions:


Autopsy sampling must include the encephalon, liver, kidneys, lungs, cardiac and peripheral blood, gastric content, bile, urine, head or body hair, and even bone. The samples collected must be stored in separated containers and filled as much as possible to minimise evaporation of volatile substances and the oxidation of drugs. The blood sample for quantitative analyses must be drawn from the femoral vein as this site is less exposed to post mortem alterations of concentrations of xenobiotics. Blood samples from different periferic sites and from the cardiac cavity, left and right, are useful for revealing the possible variation of the concentration of xenobiotics in the post-mortal period. In subjects who fall victim to fires, blood should be taken from vessels in regions spared by the fire, as the diffusion of carbon monoxide has been observed in literature. Specimens taken from different sites must be stored in different containers [5]. Urine can be taken before or during autopsy. The cerebrospinal fluid can be drawn, preferentially, by a suboccipital puncture or even aspirated from ventricles after the removal of the cranium. The gastric and intestinal content must be described in a detailed manner. Possible remains of drug tablets must be preserved in a separate container. In case subcutaneous or intravenous injection of drugs is suspected, a cutaneous sample should be taken from the site of injection and a cutaneous control sample from another region [5]. As to keratin matrices, they must preferably be natural hair cut from the posterior vertex of the head, where a lower variability of drug concentration is described [5]. The hair should be preserved at room temperature since freezing leads to a reduction of concentration. In a corpse, it is strongly advisable to take the hair sample before autopsy to avoid contamination with biological fluids, which cannot be completely removed by washings. Alternatively, sampling of pubic or axillary hairs, or nails, preferably taken from the feet, can be useful. However, nails contain lower concentrations of xenobiotics compared to hair.

In cases of corpses in an advanced state of decomposition it is necessary to collect larvae of diptera and other arthropods found on the body to perform entomotoxicological analyses, aimed at determining in the larvae the presence of xenobiotics originally present in the dead body fluids/tissues. Since living larvae rapidly metabolize the xenobiotics after removal from the corpse, they must be rapidly frozen and analysed as soon as possible. In addition, to avoid environmental contamination, the larvae must be washed before the analyses [6]. It is necessary to underline that, although larvae are useful as qualitative toxicological specimens, they seem to provide limited information of quantitive nature. Moreover the absence of xenobiotics in the larvae does not necessarily imply the absence of xenobiotics in their "food" source [6].

The toxicological analyses must encompass both qualitative and quantitative determinations, employing advanced technologies, using validated methods, foreseeing obligatory analyses of confirmation with certified reference standard of the xenobiotics identified. In general, two types of extraction procedures are foreseeable to separate and concentrate analytes from endogenous compounds: the liquid/liquid and the solid/liquid. The instrumental analyses will use advanced technologies able to separate analytes in the gaseous or liquid phase (gas

chromatography, GC, or liquid chromatography, LC) and to identify them through specific detectors such as UV, IR, FID, fluorescence, mass spectrometry and multiple mass spectrome‐ try (GC-MS, HPLC-MS, MS/MS, etc). Particularly, the use of high or low resolution mass spectrometry coupled with chromotographic technique is considered the gold standard of analyticaltechniques.Itwill alsobenecessarytodetermine concentrationratiosofparenchymahaematic, urinary-haematic, parent/metabolite compound, in order to perform a correct assessment ofresults from the historic, biological andstatistical epidemiologicalpoints of view.

**•** *general unknown research*, when it arouses suspicion of intoxication, but the substance is

**•** *specific research*, when circumstantial and/or clinical documentary evidences consent to

Autopsy sampling must include the encephalon, liver, kidneys, lungs, cardiac and peripheral blood, gastric content, bile, urine, head or body hair, and even bone. The samples collected must be stored in separated containers and filled as much as possible to minimise evaporation of volatile substances and the oxidation of drugs. The blood sample for quantitative analyses must be drawn from the femoral vein as this site is less exposed to post mortem alterations of concentrations of xenobiotics. Blood samples from different periferic sites and from the cardiac cavity, left and right, are useful for revealing the possible variation of the concentration of xenobiotics in the post-mortal period. In subjects who fall victim to fires, blood should be taken from vessels in regions spared by the fire, as the diffusion of carbon monoxide has been observed in literature. Specimens taken from different sites must be stored in different containers [5]. Urine can be taken before or during autopsy. The cerebrospinal fluid can be drawn, preferentially, by a suboccipital puncture or even aspirated from ventricles after the removal of the cranium. The gastric and intestinal content must be described in a detailed manner. Possible remains of drug tablets must be preserved in a separate container. In case subcutaneous or intravenous injection of drugs is suspected, a cutaneous sample should be taken from the site of injection and a cutaneous control sample from another region [5]. As to keratin matrices, they must preferably be natural hair cut from the posterior vertex of the head, where a lower variability of drug concentration is described [5]. The hair should be preserved at room temperature since freezing leads to a reduction of concentration. In a corpse, it is strongly advisable to take the hair sample before autopsy to avoid contamination with biological fluids, which cannot be completely removed by washings. Alternatively, sampling of pubic or axillary hairs, or nails, preferably taken from the feet, can be useful. However, nails

In cases of corpses in an advanced state of decomposition it is necessary to collect larvae of diptera and other arthropods found on the body to perform entomotoxicological analyses, aimed at determining in the larvae the presence of xenobiotics originally present in the dead body fluids/tissues. Since living larvae rapidly metabolize the xenobiotics after removal from the corpse, they must be rapidly frozen and analysed as soon as possible. In addition, to avoid environmental contamination, the larvae must be washed before the analyses [6]. It is necessary to underline that, although larvae are useful as qualitative toxicological specimens, they seem to provide limited information of quantitive nature. Moreover the absence of xenobiotics in the larvae does not necessarily imply the absence of xenobiotics in their "food" source [6]. The toxicological analyses must encompass both qualitative and quantitative determinations, employing advanced technologies, using validated methods, foreseeing obligatory analyses of confirmation with certified reference standard of the xenobiotics identified. In general, two types of extraction procedures are foreseeable to separate and concentrate analytes from endogenous compounds: the liquid/liquid and the solid/liquid. The instrumental analyses will use advanced technologies able to separate analytes in the gaseous or liquid phase (gas

hypothesise an exposure to specific substances.

88 Toxicology Studies - Cells, Drugs and Environment

contain lower concentrations of xenobiotics compared to hair.

ignored;

The laboratory of Forensic Toxicology has the primary task of assuring the accuracy of the results through processes of controls of the analytical and organizational quality. The organ‐ izational or logistic quality assurance implies the adoption of a protocol intended to preserve the chain of custody of biological specimens from the sampling, through analyses and reporting. Particular attention must be paid to the preservation procedures of the biological samples, both "in short term" (2°-4°C in the refrigerator) and "in long term" (freezer at a maximum temperature of -18°C/-20°C).

The analytical quality assurance must be achieved through the validation of methods, applying principles of selectivity, specificity, precision, accuracy and linearity. The analytical method must also be characterized by its LOD (limit of detection) and by its LOQ (limit of quantifica‐ tion). It is also necessary that the "cut-off "of the method be declared, an arbitrary measure that is adopted in order to discriminate between the results to be considered negative and the results to be considered positive. The cut-off is not therefore just a technical-analytical measure, but is also determined according to the specific diagnostic objective. The "purpose" of the research and the "aim" of the analytical element greatly influence and condition the choice of "cut off" values. Finally, it is important to remember that every analytical procedure must distinguish and appropriately use screening methods and methods of confirmation. The concept of the "confirmation" in Forensic Toxicology is indispensable, and the confirmation technique must necessarily be based on different analytical principals and/or chemicalphysical characteristics from the screening procedure.

However, a positive or negative chemical-toxicological report is not sufficient proof to affirm or exclude a death by intoxication. In the first case, for example, one might identify an insufficient concentration of the toxin, arising from accidental contact or environmental contamination. A second possible explanation can be found, for example, in the method of analysis used (high limit of detection and intrinsic limitations of the analytical technique can give an apparently negative result), or in the transformation of the toxicant in metabolites or in its elimination from the organism.

For a further exemplification, the qualitative detection of a poison in the gastrointestinal tract is not sufficient evidence to establish that the substance was the cause of death. It is necessary to demonstrate that it has been absorbed and carried through general circulation, unto the organs where it has exercised its possibly lethal effect.

Similarly, the results of the urine tests are often of little significance in determining the physiopathological effects of the toxic substance, since they only allow proof that the toxic was present in the victim's body some time before death. The physiopathological effects of the majority of the xenobiotics only correlates, in fact, with their blood concentration.

The laboratory plays a crucial role in the forensic toxicological diagnostic when a correct methodology is adopted in sampling procedures and the choice of specimen to be analysed, and when interpretation of the results is well integrated with the data acquired through other types of research.

Once the ascertainment is completed in all its stages, an evaluation phase is undertaken, in which the results of different types of tests must be comparatively and critically evaluated.

Identification of intoxication as a cause of medico-legal relevance can emerge in terms of certainty or probability, which is in turn distinguished in statistical and logical probability.

In toxicology there are not often general scientific laws, of universal or statistical epidemio‐ logical coverage, which make it possible to verify or rule out exposure to toxic substances as a cause of medico- legal relevance. It follows that one must usually have recourse to the process of rational credibility, according to the best science and experience, on the basis of what is known regarding the ethio-pathogenesis of disease from toxic origin. In the appendix, the characteristics of the most common drugs of abuse, responsible of acute or chronic intoxication, are shortly summarized according to The National Institute of Drugs of Abuse (NIDA).
