European Council of Legal Medicine (ECLM) on-site inspection forms for forensic pathology, anthropology, odontology, genetics, entomology and toxicology for forensic and medico-legal scene and corpse investigation: the Parma form.

Further to a previous publication by the European Council of Legal Medicine (ECLM) concerning on-site forensic and medico-legal scene and corpse investigation, this publication provides guidance for forensic medical specialists, pathologists and, where present, coroners' activity at a scene of death inspection and to harmonize the procedures for a correct search, detection, collection, sampling and storage of all elements which may be useful as evidence, and ensure documentation of all these steps. This ECLM's inspection form provides a checklist to be used on-site for the investigation of a corpse present at a crime or suspicious death scene. It permits the collection of all relevant data not only for the pathologist, but also for forensic anthropologists, odontologists, geneticists, entomologists and toxicologists, thus supporting a collaborative work approach. Detailed instructions for the completion of forms are provided.

European council of legal medicine (ECLM) guidelines for the examination of suspected elder abuse.

Article 25 of the Charter of Fundamental Rights of the European Union (adopted in Nice on 7 December 2000) recognizes and respects the rights of older people to lead a life of dignity and independence and to participate in social and cultural life. It also highlights the importance of prevention and recognition of elder abuse, especially since exposure to violence is likely as the population ages, either in familial or in institutional settings. Elder abuse has some issues in common with child abuse but in spite of this fact currently is less recognized. Health professionals have a major role to play in early detection and management of cases of elder abuse. This protocol summarizes some key concepts and approaches to assist in the timely detection and investigation of elder abuse cases by healthcare professionals and forensic practitioners.

Guidelines examination of victims of sexual assault harmonization of forensic and medico-legal examination of persons.

Sexual assault is a complex situation with medical, psychological, and legal aspects. Forensic experts play a major role in terms of forensic and gynecological medical examination and evidence collection in order to maintain the chain of custody. Victims should be examined by a specially trained medico-legal examiner in order to avoid multiple examinations in the surroundings that do not meet minimum health standards. The evolution and treatment of sexual assault victims are time-intensive and should optimally be provided by a team that includes a forensic medical doctor. These guidelines will be of interest to forensic medical doctors who will have responsibility for the examination and assessment of victims of sexual violence and can be used as a day-to-day service document and/or a guide to develop health service for victims of sexual violence.

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.

Obstetric litigation: the importance of the quality of clinical files and its influence on expertise conclusions.

To appreciate the Portuguese circumstances concerning situations of obstetric medico-legal conflicts and to evaluate the influence of the quality of files in expert conclusions, an analysis of all cases of obstetric medical responsibility from 2001-11 was carried out. File quality was evaluated by absence or insufficiency of clinical information supplied, by poor quality of document copies and by the registered incongruities among all the health professionals involved. Clinical files sent for forensic analysis were defective in most cases (89.5%). In about 11% of cases, expert opinion was inconclusive as a result of the poor quality of the clinical files sent for technical and scientific analysis. This situation is particularly serious in cases where the reason for the dispute was asphyxia, traumatic lesions of the newborn following instrumented delivery or shoulder dystocia and maternal sequelae, where the lack or absence of information, and poor quality copies were significantly associated with inconclusive opinions.

Bioanalytical procedures and recent developments in the determination of opiates/opioids in human biological samples.

The use and abuse of illegal drugs affects all modern societies, and therefore the assessment of drug exposure is an important task that needs to be accomplished. For this reason, the reliable determination of these drugs and their metabolites in biological specimens is an issue of utmost relevance for both clinical and forensic toxicology laboratories in their fields of expertise, including in utero drug exposure, driving under the influence of drugs and drug use in workplace scenarios. Most of the confirmatory analyses for abused drugs in biological samples are performed by gas chromatographic-mass spectrometric methods, but use of the more recent and sensitive liquid chromatography-(tandem) mass spectrometry technology is increasing dramatically. This article reviews recently published articles that describe procedures for the detection of opiates in the most commonly used human biological matrices, blood and urine, and also in unconventional ones, e.g. oral fluid, hair, and meconium. Special attention will be paid to sample preparation and chromatographic analysis.

Simultaneous quantitation of morphine, 6-acetylmorphine, codeine, 6-acetylcodeine and tramadol in hair using mixed-mode solid-phase extraction and gas chromatography-mass spectrometry.

A simple procedure has been developed and validated for the qualitative and quantitative analysis of several opiates (morphine, 6-acetylmorphine, codeine, 6-acetylcodeine) and tramadol in hair. The analytes were extracted from within the matrix via an overnight incubation with methanol at 65 degrees C, and afterwards the samples were cleaned up by mixed-mode solid-phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide with 5% trimethylchlorosilane and analyzed by gas chromatography-mass spectrometry in the selected ion monitoring mode. The method was linear from 0.05 (lower limit of quantitation) to 50 ng/mg (40 ng/mg for tramadol), with correlation coefficients higher than 0.99 for all compounds, accomplishing the cut-off values proposed by the Society of Hair Testing for the detection of these substances in hair (0.2 ng/mg). Intra- and interday precision and trueness were in conformity with the criteria normally accepted in bioanalytical method validation, and the sample cleanup step presented a mean efficiency higher than 90% for all analytes. Furthermore, using these incubation conditions, 6-acetylmorphine did not significantly hydrolyze to morphine. For these reasons, and because of its simplicity, the proposed method can be successfully applied in the determination of these compounds in hair samples, and is suitable for application in routine analysis with forensic purposes.

Mixed-mode solid-phase extraction for sample cleanup in hair analysis for methadone and its main metabolite.

A simple and rapid method for the determination of methadone and its main metabolite EDDP in hair has been developed and validated. The analytes were completely extracted from the matrix after a short alkaline incubation, and the extracts were further cleaned up by solid-phase extraction using mixed-mode cartridges. Linearity was obtained from 0.1 (lower limit of quantitation, LLOQ) to 30 ng/mg for both compounds, with correlation coefficients higher than 0.99. Intra- and interday precision and accuracy were in conformity with internationally accepted guidelines for bioanalytical method validation, and the cleanup procedure presented mean extraction efficiencies higher than 90% for both analytes. This high efficiency greatly contributed to the low limits of quantitation achieved, and therefore this method can be successfully applied in the determination of methadone and EDDP in hair samples in clinical and forensic scenarios where these compounds are involved.

Microextraction by Packed Sorbent as a Novel Strategy for Sample Clean-Up in the Determination of Methadone and EDDP in Hair.

A microextraction by packed sorbent (MEPS) procedure for rapid concentration of methadone and its primary metabolite (EDDP) in hair samples was developed. The miniaturized approach coupled to gas chromatography with tandem mass spectrometry (GC-MS-MS) was successfully validated. Hair samples (50 mg) were incubated with 1 mL of 1 M sodium hydroxide for 45 min at 50°C, time after which the extract was neutralized by adding 100 µL of 20% formic acid. Subsequently, MEPS was applied using a M1 sorbent (4 mg; 80% C8 and 20% strong cation-exchange (SCX)), first conditioned with three 250-µL cycles of methanol and three 250-µL cycles of 2% formic acid. The extract load occurred with nine 150-µL cycles followed by a washing step involving three 50-µL cycles with 3.36% formic acid. For the elution of the analytes, six 100-µL cycles of 2.36% ammonium hydroxide in methanol were applied. The method was linear from 0.01 to 5 ng/mg, for both compounds, presenting determination coefficients greater than 0.99. Precision and accuracy were in accordance with the statements of international guidelines for method validation. This new miniaturized approach allowed obtaining recoveries ranging from 73 to 109% for methadone and 84 to 110% for EDDP, proving to be an excellent alternative to classic approaches, as well as other miniaturized procedures.

Solid-phase extraction and gas chromatographic- mass spectrometric determination of the veterinary drug xylazine in human blood.

This paper presents a method for the determination of xylazine in whole blood using solid-phase extraction and gas chromatography-mass spectrometry. This technique required only 0.5 mL of sample, and protriptyline was used as internal standard (IS). Limits of detection and quantitation (LOQ) were 2 and 10 ng/mL, respectively. The method was found to be linear between the LOQ and 3.50 microg/mL, with correlation coefficients higher than 0.9922. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The analyte was stable in the matrix for at least 18 h at room temperature and for at least three freeze/thaw cycles. Mean recovery, calculated at three concentration levels, was 87%. To the best of our knowledge, this is the first time that solid-phase extraction is used as sample preparation technique for the determination of this compound in biological media. Because of its simplicity and speed when compared to other extraction techniques, the herein described method can be successfully applied in the diagnosis of intoxications by xylazine.

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry.

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.

Analytical approach to determine ticlopidine in post-mortem blood.

A new and sensitive method to determine ticlopidine in whole human blood using proadifen as the internal standard (IS) is described. The analyte and IS were extracted by solid-phase extraction using Oasis HLB cartridges, and the extracts were analyzed by gas chromatography-electron impact ionisation-mass spectrometry (GC/EI-MS). Calibration curves were established daily in spiked blood samples using a non-linear calibration model, between 0.01 and 4.5 microg/mL. The correlation coefficients were higher than 0.995. Precision and accuracy fulfilled the internationally accepted criteria (coefficients of variation were less than 9%, and the measured concentrations were within +/-7% of the true value). Limits of detection and quantitation were respectively, 3 and 10 ng/mL. No interfering substances were detected by analysis of 10 blank blood samples of different origin. Mean recovery, calculated at three concentration levels, was 71%. Because of its simplicity and speed, the proposed method can be applied in the determination of this inhibitor of platelet aggregation in whole blood samples, and is suitable for application in toxicology routine analysis.

Application of solid phase microextraction to the determination of strychnine in blood.

A simple and rapid method based on solid phase microextraction (SPME) via direct immersion followed by gas chromatography coupled with electron impact ionization/mass spectrometry (GC/EI-MS) was developed for the determination of strychnine in blood. Papaverine was used as internal standard (I.S.). Two types of fibre coating were tested, 100 microm polydimethylsiloxane and 65 microm Carbowax/Divinylbenzene, the latter giving higher recoveries of the compound. The main factors affecting the SPME process, such as sample dilution (1:10), adsorption and desorption times (20 and 10 min, respectively), carry-over effect (not observed), pH and salt addition (no modifications on pH or salt concentration) were optimized. The procedure was validated in terms of linearity (r(2)=0.9992 for concentrations ranging from 0.10 to 5.00 microg/mL), intra and interday precision (0.93 and 4.62%, respectively at 0.50 microg/mL; 3.33 and 8.06%, respectively at 2.50 microg/mL), sensitivity (6.83 and 8.91 ng/mL for LOD and LOQ, respectively) and extraction recovery (0.54 and 0.39% at 0.50 and 2.50 microg/mL, respectively). The developed procedure was found suitable for forensic investigations and was considered a good alternative to the liquid-liquid extraction methods normally used for the determination of this compound in biological media.

Population data on 15 autosomal STRs in a sample from East Timor.

Allele frequencies for the fifteen STRs included in the AmpF/STR Identifiler (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPO and VWA) were estimated from a sample of 186 unrelated individuals from East Timor. No deviations from Hardy-Weinberg equilibrium were observed (only after applying the Bonferroni correction in the cases of D2S1338, TPO and D5S818). Genetic parameters of forensic interest were calculated and comparison with geographically nearby populations was performed.

Determination of strychnine in human blood using solid-phase extraction and GC-EI-MS.

A rapid, simple, and sensitive method has been developed for the identification and quantitation of strychnine in human blood. The sample cleanup procedure involved solid-phase extraction with Oasis(R) HLB cartridges. The extracts were analyzed by gas chromatography-electron impact ionization-mass spectrometry. Limits of detection (LOD) and quantitation (LOQ) were 0.03 and 0.10 microg/mL, respectively, and the method was found to be linear between the LOQ and 2.5 microg/mL, with a correlation coefficient of 0.9994. Intra- and interday precision and accuracy were determined at both low and high concentrations (0.50 and 2.00 microg/mL). The CVs ranged from 5.63 to 8.50% and bias was within +/- 10% of the true value. Mean recovery of strychnine was 90.7%. Because of its simplicity and speed, the described method can be applied in forensic toxicology laboratories to determine this alkaloid in whole blood samples. Also, the fact that only 0.5 mL of blood is required to accomplish the analysis make this procedure useful in situations where several exams are needed and the sample volume is limited.

Biochemical assessment of acute myocardial ischaemia.

AIMS: To evaluate the efficacy of biochemical parameters in different fluids in the diagnosis of myocardial infarction of different causes, analysed after death. METHODS: The myoglobin concentration and total creatine kinase (CK) and creatine kinase MB isoenzyme (CK-MB) activities were measured in serum, pericardial fluid, and vitreous humour from seven diagnostic groups of cadavers classified according to the severity of myocardial ischaemia and cause of death. Lactate dehydrogenase (LDH) and myosin were measured only in serum and pericardial fluid, and cathepsin D only in pericardial fluid. Routine haematoxylin and eosin and acridine orange staining were used for microscopy studies of heart tissue. RESULTS: In pericardial fluid there were substantial differences between the different groups with respect to CK, CK-MB, and LDH activities and myosin concentrations. The highest values were found in cases with morphological evidence of myocardial ischaemia. CONCLUSIONS: Biochemical parameters, which reach the pericardial fluid via passive diffusion and ultrafiltration due to a pressure gradient, were thus detectable in this fluid earlier than in serum in cases with myocardial ischaemia. These biochemical parameters may be of use for ruling out myocardial ischaemia in those controversial cases in which reliable morphological findings are lacking.

A fatal forensic intoxication with fenarimol: analysis by HPLC/DAD/MSD.

Fenarimol (Rubigan) is a pyrimidine ergosterol biosynthesis inhibitor used as a systemic fungicide. The authors present a fatal fenarimol intoxication case analysed in the Forensic Toxicology Service of the National Institute of Legal Medicine. The results were used to compare two different HPLC techniques, regarding selectivity and sensitivity: an HPLC system with a diode array detector (DAD) and an HPLC system with a DAD and a mass spectrometry detector (MSD) with an electrospray interface. All biological samples were submitted to a solid-phase extraction procedure. The detection and quantification limits of fenarimol, linearity, precision and accuracy were evaluated. The fenarimol concentration levels determined were of 89.0 mg/ml in gastric contents, 1.9 mg/g in liver and 0.4 mg/g in kidney. Blood was not available at autopsy. No published data related to fenarimol self-poisoning were found, so it was not possible to interpret the results obtained by comparison with toxic/lethal levels.

Diagnostic ability of D-dimer in the establishment of the vitality of wounds.

The potential of skin measurements of D-dimer (DD), a specific derivative of crosslinked fibrin, for the diagnosis of the vital origin of wounds was investigated in 67 human vital skin wounds and homolateral control pieces of skin from 53 autopsies performed at the Institute of Legal Medicine of Coimbra (Portugal) during 1992. Our results demonstrate that DD levels for the incised vital skin wounds (n = 50) were much higher on average than their controls (P < 0.01) which were actually incised injuries produced postmortem at the autopsy. We did not find any statistical difference in average DD levels among the remaining injuries (seven abrasions and ten contusions) in comparison to their respective controls. DD may be used, with certain limitations, as a marker of the vitality of incised wounds. The authors believe this is the first time this marker has been applied in forensic pathology. It is an example of an easy and quick application technique, with the added advantage of low cost.

Analytical method for the determination of strychnine in tissues by gas chromatography/mass spectrometry: two case reports.

This paper describes an analytical method for strychnine determination in biological samples by gas chromatography/mass spectrometry and their application in the investigation of two cases involving strychnine ingestion: A fatal case and a clinical one. The strychnine is isolated from biological samples using a liquid-liquid extraction procedure. The clean-up procedure is performed using an acid solution. Papaverine is used as internal standard in the quantification of strychnine. In the analysed specimens, the limits of quantification were 0.1 microg/ml or 0.1 microg/g. The recovery rate ranged from 75.0% to 98.7% and the coefficients of variation ranged from 4.8% to 10.5%.

Population distribution of six PCR-amplified loci in Madeira Archipelago (Portugal).

Frequency data of the short tandem repeat (STR) loci HUMTH01, HUMVWA31/A, HUMF13A1, HUMFES/FPS, D12S391 and HUMFIBRA/FGA were determined in blood stains obtained from a population of unrelated individuals from the Madeira Archipelago. The observed genotype distribution showed no significant deviation from the Hardy-Weinberg equilibrium and there was no evidence for association of alleles among the six loci. Population data showed a combined discrimination power of 0.9999998 and a chance of exclusion of 0.99597. The frequencies are similar to those of other compared caucasian populations but significant differences were found between the Madeira population and Japanese, Chinese, Greenland Eskimos and Quechua Amerindians. The six loci studied, together proved to be highly discriminating and valuable for forensic cases.