European Council of Legal Medicine (ECLM) principles for on-site forensic and medico-legal scene and corpse investigation.

Forensic medical practitioners need to define the general principles governing procedures to be used for the on-site examination of a body where the death has occurred in unnatural, violent or suspicious circumstances. These principles should be followed whenever a medical expert is required to perform an on-site corpse inspection and should be utilised as a set of general guidelines to be adapted to the specific situation in hand and interpreted using common sense and scientific knowledge of the relevant procedures and facts of the case. The aim of these principles is to ensure that forensic evidence at the scene of a death is properly observed and assessed and all necessary relevant evidence gathered in order to ensure that a comprehensive report is available to the judicial authority (investigating judge or coroner) in the justice system. The on-site corpse inspection by a forensic practitioner is a mandatory and essential stage of the forensic and medico-legal autopsy, as it may provide important information for subsequent investigation stages.

Whiplash-Associated Disorders : Clinical and medico-legal guidelines on the methods of ascertainment.

The manuscript presents the International Guidelines developed by the Working Group on Personal Injury and Damage under the patronage of the International Academy of Legal Medicine (IALM) regarding the Methods of Ascertainment of any suspected Whiplash-Associated Disorders (WAD).The document includes a detailed description of the logical and methodological steps of the ascertainment process as well as a synoptic diagram in the form of Flow Chart.

Psychic trauma as cause of death.

AIM: of study Psychic trauma is described as the action of 'an emotionally overwhelming factor' capable of causing neurovegetative alterations leading to transitory or persisting bodily changes. The medico-legal concept of psychic trauma and its definition as a cause in penal cases is debated. The authors present three cases of death after psychic trauma, and discuss the definition of cause within the penal ambit of identified 'emotionally overwhelming factors'. MATERIALS AND METHODS: The methodological approach to ascertainment and criterion-based assessment in each case involved the following phases: (1) examination of circumstantial evidence, clinical records and documentation; (2) autopsy; (3) ascertainment of cause of death; and (4) ascertainment of psychic trauma, and its coexisting relationship with the cause of death. RESULTS: The results and assessment of each of the three cases are discussed from the viewpoint of the causal connotation of psychic trauma. In the cases presented, psychic trauma caused death, as deduced from assessment of the type of externally caused emotional insult, the subjects' personal characteristics and the circumstances of the event causing death. CONCLUSIONS: In cases of death due to psychic trauma, careful methodological ascertainment is essential, with the double aim of defining 'emotionally overwhelming factors' as a significant cause of death from the penal point of view, and of identifying the responsibility of third parties involved in the death event and associated dynamics of homicide.

Forensic application of ESEM and XRF-EDS techniques to a fatal case of sodium phosphate enema intoxication.

Sodium phosphate enemas and laxatives are widely used for the treatment of constipation, even if a number of cases of significant toxicity due to alterations of the fluid and electrolyte equilibria (hypernatremia, hyperphosphatemia, and hypocalcemia) have been reported. We present the case of an 83-year-old man who died of fecal and chemical peritonitis secondary to an iatrogenic colon perforation (produced performing a Fleet enema through the patient's iliac colostomy) with peritoneal absorption of sodium phosphate. Environmental scanning electron microscopy coupled with an X-ray fluorescence energy dispersive spectrometry discovered multiple bright crystals formed of calcium, phosphorus, and oxygen in the brain, heart, lung, and kidney sections of the victim. The absence of these kinds of precipitates in two control samples chronically treated with Fleet enemas led us to assume that the deceased had adsorbed a great quantity of phosphorus ions from the peritoneal cavity with subsequent systemic dissemination and precipitation of calcium phosphate bindings.

Use of Lucilia species for forensic investigations in Southern Europe.

The aim of this study was to highlight the importance of evaluating entomological evidence in forensic investigations on a regional scale. To evaluate climatic, geographical and environmental influences on the selection of carrion-breeding fauna in Northern Italy and consequently on inferred forensic data (post-mortem intervals and post-mortem transfer), we present details of six indoor-outdoor cases. Results show that the most abundant species was Lucilia sericata, together with other fly species of entomo-forensic interest, belonging to the Calliphoridae and Sarcophagidae families. In particular, for the first time in Italy, we report finding Phormia regina, Lucilia ampullacea, Lucilia caesar and Sarcophaga (Pandelleana) protuberans on fresh cadavers. The active period of L. sericata in Northern Italy, according to previous findings in Southern Europe, revealing clearcut differences with phenologies in Northern Europe, has important consequences in estimating the period (season, months) of death in cases of long post-mortem intervals (several months or years) if empty puparia of this fly are found. According to our results, the distribution of L. sericata in areas with urban sprawl, like Northern Italian regions, cannot be used to evaluate post-mortem transfer from an urban area to a rural one.

Selective stroke of the solitary tract nuclei in two cases of central sleep apnoea.

Central sleep apnoea (CSA) is a breathing disorder characterized by repetitive central apnoeas with hypoxia interrupted by hyperventilation phases. In the literature, there are reports of CSA caused by brainstem infarcts. We report two patients (38 and 53 years old) with longstanding history of central sleep apnoea who died during sleep. In both cases the autopsy revealed acute bilateral hypoxic lesions at the level of the solitary tract nuclei. In one case, symmetrical selective neuronal necrosis was found in the dorsal part of the solitary tract nuclei. A chronic obstructive vasculopathy was also found, with thickening and fibrosis of the smallest vessels of the medullary tegmentum. In the other case, bilateral infarctions were found with the base at the ependymal lining of the 4th ventricle floor and the apex towards the solitary tract. An acute intramural hemorrhagic lesion in the premedullary segment of the left vertebral artery was also found. Episodes of hypoxemic hypoxia during sleep may worsen the effects of focal oligohemic hypoxia in the medullary tegmentum. Selective stroke of the solitary tract nuclei may be the acute fatal lesion in patients with both central sleep apnoea and lesions of the vertebro-basilar system. To the best of our knowledge, this is the first neuropathologic report of acute medullary ischemic-hypoxic lesions which may not be considered the cause of the CSA because of their recent onset. Our findings suggest that CSA, besides being caused by ischemic events at the level of the medulla, may also contribute to pathogenesis of strokes, through hypoxia or hemodynamic oscillations.

Histopathology of carotid body in heroin addiction. Possible chemosensitive impairment.

AIMS: To perform a morphometric analysis of carotid bodies in opiate addicts. METHODS AND RESULTS: Carotid bodies were sampled at autopsy from 35 subjects who died of heroin intoxication (mean age 26 years), and from eight young (22 years) and eight older subjects (66.5 years) who died of trauma. Sections were stained with haematoxylin-eosin, azan-Mallory, and double-labelling immunohistochemistry with antineuronal specific enolase and anti-S100, to count type I and type II cells. Interlobular and intralobular connective tissue was increased both in the opiate cases (43.45 +/- 6.79%, P < 0.001, and 13.34 +/- 5.72%, P < 0.001, respectively) and older cases (46.67 +/- 1.65%, P < 0.001, and 9.62 +/- 2.11%, P < 0.05, respectively) compared with young cases (33.17 +/- 6.41% and 4.33 +/- 1.84%, respectively). The percentage of type II cells in the opiate cases (51.6 +/- 7.3%, P < 0.001) and in the older controls (49.0 +/- 7.2%, P < 0.01) was higher than in the young cases (37.9 +/- 3.0%). Among type I cells, the light cell percentage in the opiate cases (65.85 +/- 11%, P < 0.001) was reduced with respect to the two control groups (82.8 +/- 5.34%, young; 81.62 +/- 8.58%, older). CONCLUSIONS: The increases in connective tissue and type II cells are similar to findings in ageing and chronic pulmonary disease, and may be ascribed to glomic hypoxia. A direct action of opiates should be taken into account for the decrease in light cells in heroin addiction. The histopathological changes in the carotid body, by impairing chemosensivity, may play a role in the fatal cardiorespiratory derangement of heroin addicts.

Fatal toxic epidermal necrolysis in autoimmune polyglandular syndrome type I.

Autoimmune polyglandular syndrome Type I (APS I) is a disorder defined by the presence of at least two of the following diseases: Addison's disease, hypoparathyroidism, and chronic mucocutaneous candidiasis. We present the case of a 45-yr-old woman, affected by APS I, in chronic treatment with betamethasone. She was referred to a Division of General Medicine for jaundice, ascites and peripheral edema attributed to worsening of pre-existing autoimmune chronic hepatitis. During hospitalization, the following drugs were given: Amoxicillin/Clavulanic acid and Levofloxacin for bronchopneumonia, Furosemide and Canreonate for renal impairment, Pantoprazole for gastric protection, and Itraconazole for oral candidiasis. After about a month, she developed widespread, sheet-like, epidermal detachment, with painful lesions of the conjunctiva, lips and mouth. Toxic epidermal necrolysis (TEN) was diagnosed, and the patient was transferred to a Burn Center, where she died 10 days after the first onset of cutaneous rash. Autoptic and histopathological findings (epidermal necrosis and detachment, lymphomonocytic infiltration of the dermis) confirmed the clinical diagnosis. TEN is a usually drug-induced cutaneous inflammatory disorder characterized by extensive epidermal detachment and frequent mucosal involvement. It has also been associated with immuno-mediated disorders (HIV infection, graft-vs-host disease, systemic lupus erythematosus, mixed essential cryoglobulinemia), in keeping with immuno-mediated pathogenesis. We present, to our knowledge, the first report of TEN in a patient with APS I, and suggest that some pathogenetic mechanisms of APS I are shared with TEN. We stress how such a disease can occur in an autoimmune syndrome, even during corticosteroid treatment.

Determination of midazolam in human plasma by solid-phase microextraction and gas chromatography/mass spectrometry.

A new method is described for the qualitative and quantitative analysis of midazolam, a short-acting 1,4-imidazole benzodiazepine, in human plasma. It involves a plasma deproteinization step, solid-phase microextraction (SPME) of midazolam using an 85-microm polyacrylate fiber, and its detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode, using pinazepam as internal standard. The assay is linear over a midazolam plasma range of 1.5-300 ng/mL, relative intra- and inter-assay standard deviations at 5 ng/mL are below 7%, and the limit of detection is 1 ng/mL. The method is simple, fast and sufficiently sensitive to be applied in clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.

[Medico-legal features of early discharge in acute myocardial infarction and chest pain]. / Aspetti medico-legali della dimissione precoce nell'infarto miocardico acuto e nel dolore toracico

The authors' aim is to outline some of the main medico-legal problems in cardiology, especially those regarding the premature hospital discharge of patients with undefined chest pain and/or with acute myocardial infarction. After a brief overview on the etiology and clinical definition of chest pain and myocardial infarction, premature hospital discharge is defined and the incidental medico-legal risks that physicians operating in such situations are exposed to are pointed out. Next, the profiles regarding both the positive and negative views of professional medical responsibility are described. In the negative frame, the authors outline the most frequent civil and penal aspects of the unpremeditated responsibility. Then the physician's error, in both qualitative (generic or specific guilt) and quantitative (degree) terms, is considered; particularly, negligence, imprudence and inexperience, as qualitatively accepted meanings of generic guilt, are dealt with by adopting illustrative cases settled in the light of the right legal interpretation. The phases of the diagnostic or prognostic error are evaluated, and clinical protocols, as a reference parameter for the identification of error, are considered. Lastly, the problem of causality, essential condition for the judgment about the professional responsibility, and the problem of the patient's consent, including an evaluation of the legal capability or incapability about the declaration of consent, are examined closely.

Determination of gamma-hydroxybutyric acid (GHB) in plasma and urine by headspace solid-phase microextraction and gas chromatography/positive ion chemical ionization mass spectrometry.

A new method for the qualitative and quantitative analysis of gamma-hydroxybutyric acid (GHB) in plasma and urine samples is described. It involves the conversion of GHB to gamma-butyrolactone (GBL), its subsequent headspace solid-phase microextraction (SPME), and detection by gas chromatography/positive ion chemical ionization mass spectrometry (GC/PICI-MS), using D(6)-GBL as internal standard. The assay is linear over a plasma GHB range of 1-100 microg/mL (n = 5, r = 0.999) and a urine GHB range of 5-150 microg/mL (n = 5, r = 0. 998). Relative intra- and inter-assay standard deviations, determined for plasma and urine samples at 5 and 50 microg/mL, are all below 5%. The method is simple, specific and reasonably fast. It may be applied for clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.

[Alcohol, drugs, pharmacologic agents, and street safety]. / Alcol, droga, farmaci e sicurezza stradale.

Deaths due to road accidents during weekends have become a worrying phenomenon in Italy. With the aim of highlighting the role of psychotropic substances (alcohol, drugs of abuse) in causing road accidents, a survey based on clinical and chemico-toxicological analyses has been carried out on car drivers in the Veneto region during night weekends since 1994. Rapid clinical screening was carried out on 7952 drivers. 1399 of these, suspected to be under the influence of psychotropic substances, were subjected to complete clinical and toxicological ascertainment involving the following procedures: a) anamnesis, aiming at evidence of possible current or past use of psychotropic substances; b) objective clinical examination, aiming at finding evidence of recent (signs of acute or chronic intoxication) or past use (signs of withdrawal or associated organic pathologies) of psychotropic substances; c) double sampling of blood and urine and chemico-toxicological analysis using immunochemical, GC-HS and GC/MS-SIM techniques. As well as many data of social and behavioural interest, processing of results demonstrated that: a) 56.7% of the drivers examined had consumed alcoholic beverages; b) 30.4% had BACs higher than the threshold permitted in Italy (80 mg/100 ml); c) 15.7% of drivers were found to be under the influence of drugs of abuse or psychoactive drugs; d) the most frequently found substances were (in order): cannabinoids, stimulants (cocaine, amphetamines), opiates.

Effects of single dose of gamma-hydroxybutyric acid and lorazepam on psychomotor performance and subjective feelings in healthy volunteers.

OBJECTIVE: This study investigated the possible effects of gamma-hydroxybutyric acid (GHB) on human psychomotor performance and subjective feelings important for the safety of skilled performance. METHODS: Twelve healthy volunteers, six males and six females, aged 22-36 years, participated as subjects. Drugs and placebo were administered according to a single-dose, double-blind, balanced, four-way, crossover design. Treatments were separated by a wash-out period of 1 week and consisted of placebo, lorazepam 0.03 mg x kg(-1), GHB 12.5 mg x kg(-1) and GHB 25 mg x kg(-1). Subjects' psychomotor performance was assessed at baseline and at 15, 60, 120 and 180 min after treatment. Mood was assessed using 16 visual analogue scales, before treatment and 120 min later. Psychomotor performance was measured using the following tests: Critical Flicker Fusion. Response Competition Test, Critical Tracking Task, Choice Reaction Time and Visual Vigilance Task. RESULTS: GHB at both doses had no effects on attention, vigilance, alertness, short-term memory or psychomotor co-ordination (delta-placebo, P > 0.05); calmness increased with the lower dose and contentedness decreased significantly at both doses (delta-baseline, P < 0.05); adverse effects were limited to slight subjective feelings of dizziness and dullness, which disappeared 30-60 min after administration of the dose. Lorazepam caused impairment of psychometric functions. CONCLUSION: After single therapeutic doses, GHB does not induce changes in psychomotor performance and therefore the drug does not influence the ability to drive or work. However, repeated reports of the abuse potential of GHB and its usefulness in treating ethyl alcohol addiction indicate that it may play an "agonist-like" role, which means that it should only used under close medical supervision.

Proficiency testing for psychoactive substances in Italy.

This paper describes the general design and main results of the Italian proficiency testing program for the analysis of psychoactive substances in urine, a long-term initiative created in 1995 on an educational basis and characterized by an innovative internet-based service for data exchange between laboratories and the organizing body. Batches of six urine samples, validated by reference laboratories, are sent every 3 months to participating laboratories, which may choose which classes of substances to test from those planned by the program panel and, within those classes, which type of analytical commitment to work on: identification of just one class (Option 1), identification of single substances (Option 2), or identification and quantification of single substances (Option 3). Comprehensive periodical reports and annual reports are provided to participants with evaluation of their performance and an annual workshop is organized to discuss technical-scientific topics related to clinical, forensic and analytical toxicology. About 200 laboratories currently participate in the program and a total of 67,059 analyses have been carried out since 1995. The mean percentage of correct results was 96.8%, with a yearly improvement of about 0.4%. The best average false positive and false negative rates were obtained for methadone (0.2% and 2.1% respectively) and cocaine (0.3% and 2.2%). The worst average false positive rates were obtained for amphetamines and opiates (3.2% and 5.0%) and worst average false negative rates for amphetamines, barbiturates and cannabinoids (17.4%, 30.7% and 19.9%).

Opiate-related death: morphometric study of neurons from the dorsal motor nucleus of the vagus nerve.

The aim of this work is to verify the presence of objectively observable morphological alterations caused by the direct action of opiates on brain structures controlling respiration in cases of drug-related deaths. A computer-assisted morphometric study was carried out on the Dorsal Motor Nucleus of the Vagus Nerve (DMNV) in seven subjects (four of whom died of drug overdose, and 3 controls). Serial sections 10-15 microns thick were stained according to Nissl. The sections were then examined under the optical microscope and 200 fields per subject were digitalized. The resulting images, regarding at least 700 neurons per subject were segmented using a semi-automatic procedure and analysed by the Optilab software. The data obtained were subjected to cluster analysis to identify the neuron classes in the DMNV. Each subject was characterized with a vector of percentages indicating the fraction of neurons falling in each of these classes. Discriminant analysis was then applied to verify to what extent the variables discriminated between the two populations. Results indicated that: the statistical methods adopted have a good power of discrimination; in subjects who died of opiate overdose, the neurons are distributed to a greater extent in classes composed of smaller neurons than in controls.

Effect of moderate or severe liver dysfunction on the pharmacokinetics of gamma-hydroxybutyric acid.

OBJECTIVES: To assess the effect of moderate or severe liver dysfunction on the pharmacokinetics of gamma-hydroxybutyric acid (GHB). METHODS: The absorption and disposition kinetics of GHB were studied in eight cirrhotic patients without ascites (Child's class A) and eight cirrhotic patients with ascites (Child's class C), after administration of a single oral dose of 25 mg.kg-1. The liver metabolic function of each patient was evaluated by measuring antipyrine clearance and the formation rate of the lidocaine metabolite monoethylglycinexylidide (MEGX). RESULTS: Compared to those previously determined in eight healthy control subjects given the same GHB dose, mean AUC values were double or greater in the cirrhotic patients. Accordingly, apparent oral clearance was markedly reduced (from 9.1 to 4.5 and 4.1 ml.min-1.kg-1 in nonascitic and ascitic patients, respectively). Terminal half-life (t1/2), was significantly longer in nonascitic patients than in control subjects (32 vs 22 min). A further significant prolongation of t1/2, most likely due to an increased distribution volume, was observed in patients with ascites (56 min). Nonetheless, GHB plasma concentrations fell to either undetectable or negligible levels by the end of the usual dosing intervals (6-8 h). More limited changes were noted in the absorption parameters. The peak level (Cmax) increased only in nonascitic patients, but not proportionally to the increase in AUC. The time to Cmax increased from 30 to 45 min in both cirrhotic groups. These findings are consistent with a slowed rate of GHB absorption in cirrhotic patients. Adverse effects were similar, for intensity and duration, to those recorded in healthy volunteers, i.e., mild and transient. CONCLUSIONS: Although liver cirrhosis causes significant modifications of GHB disposition kinetics, the increase in t1/2 is not such as to cause drug accumulation on repetitive dosing. However, in consideration of the higher mean plasma levels observed in cirrhotic patients, it appears wise to keep the initial GHB daily dose at the lower end of the therapeutic range and to carefully monitor the patients if upward dose adjustments are required.

Fatality due to gamma-hydroxybutyric acid (GHB) and heroin intoxication.

The first case of fatal intoxication due to ingestion of gamma-hydroxybutyric acid (GHB) and intravenous use of heroin is reported. A 42-year-old man, known to have been a heroin addict and to have taken other psychoactive substances, who had been in treatment with GHB for several months, was found dead. Anatomohistopathologic examination showed generalized visceral congestion, edema and pulmonary anthracosis, chronic bronchitis and chronic active hepatitis. Toxicological findings included fluid and tissue distributions of GHB, morphine and 6-monoacetylmorphine. GHB and morphine concentrations were respectively 11.5 and 0.77 micrograms/mL (blood), 84.3 and 0.3 micrograms/mL (vitreous humor), 258.3 and 1.35 micrograms/mL (urine), 57.0 and 14.3 micrograms/mL (bile), 40.0 and 0.43 micrograms/g (brain), 43.0 and 0.60 micrograms/g (liver), 47.0 and 0.68 micrograms/g (kidney). Blood and urine levels of 6-monoacetylmorphine were 28.5 and 12.1 ng/mL respectively. The presumed mechanism of action and pharmacokinetics of GHB are briefly reviewed, with reference to its therapeutic use and to reports of non-fatal GHB intoxication.

PCR analysis of the short tandem repeat (STR) system HUMVWA31. Allele and genotype frequencies in an Italian population sample.

A population study in a sample of 211 unrelated individuals from 2 cities in North and Central Italy was carried out to investigate the short tandem repeat (STR) system HUMVWA31. Separation of PCR- amplified DNA fragments was performed by high-resolution horizontal denaturing polyacrylamide gel electrophoresis (PAGE) followed by silver staining. The 7 common alleles were found, together with a new smaller allele. Distribution of the observed genotypes did not deviate from Hardy-Weinberg (H-W) equilibrium. The power of discrimination for this locus was 0.93 and the chance of exclusion was 0.61. Good agreement was found between the allele frequencies in 2 Italian population samples and previous studies on Caucasians. The results of this study suggest that this STR system may be a useful tool in forensic investigations.

Diagnosis of HIV infection from bloodstains by PCR. A further marker for identification.

The forensic usefulness of the detection of HIV infection in bloodstains is linked to the increasing spread of HIV infection and the consequent rise in the number of forensic cases involving HIV-positive subjects. This study was designed to detect HIV infection in bloodstains of various ages obtained from HIV-positive patients treated with zidovudine (3'-azido-3'-deoxythymidine, AZT) using PCR methods. Of the 3 kinds of extraction and amplification methods checked, only a nested PCR method for the amplification of a sequence located in the HIV Reverse Transcriptase (RT) region was successful. This method, involving 2 amplification steps (1: fragment of 768 bp; 2: codons 41-67-70-215-219), encompasses the mutations commonly observed during AZT therapy and overcomes the limitations inherent in serological testing. The discriminatory power of the method can detect specific mutation patterns in the RT gene linked to drug resistance and compares the specific pattern of the blood-stain with fresh blood or other specimens from the subject in question.

Drugs-of-abuse testing in urine: statistical approach and experimental comparison of immunochemical and chromatographic techniques.

This study deals with the experimental and statistical comparison of six immunochemical techniques, including noninstrumental on-site and instrumental formats (EIA-EMIT and EZ-SCREEN; FPIA-ADx; RIA-Coat-A-Count; LI-Abuscreen ONTRAK; CBI-Triage), and three chromatographic techniques (TLC-Toxi-Lab; HPLC; HPLC-REMEDi drug profiling system), using GC-MS as a reference technique for analyzing amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, methadone, and opiates in the urine of various kinds of drug users. The study reports (a) the values of sensitivity, specificity, false-positive rates, and false-negative rates of each technique; (b) the results of bayesian statistical analysis, which are based on prevalence values of the samples examined and expressed as positive and negative predictive values and cumulative predictive values for each single technique and for combinations of paired immunochemical and chromatographic techniques; and (c) the results of a rough classification of the various degrees of predictability of these techniques. Lastly, this study proposes a decision-making process for establishing the best combination of analytical techniques for the goals in question, according to the characteristics and facilities of each laboratory.