New miniaturized clean-up procedure for hair samples by means of microextraction by packed sorbent: determination of cocaine and metabolites.

Cocaine is still one of the most commonly used illicit substances worldwide, with an estimated 4 million users in Europe in the last year. Hair samples have been widely used for the determination of episodic or repeated consumption of this substance, but the use of miniaturized techniques for hair sample clean-up has been challenging due to the sample complexity. Despite hair's complex matrix, MEPS provides a method that is fast, reduces the volume of extraction solvents used, and offers low-cost options (since extraction beds may be reused several times). Microextraction by packed sorbent using a mixed-mode sorbent was optimized for hair sample clean-up in order to determine cocaine, benzoylecgonine, ecgonine methyl ester, norcocaine, cocaethylene and anhydroecgonine methyl ester by gas chromatography coupled to tandem mass spectrometry. The method was fully validated according to internationally accepted criteria, presenting good linearity between the limits of quantification (0.01-0.15) and 5 ng/mg. Precision and accuracy resulted in coefficients of variation typically lower than 15%, with mean relative errors within ±15% for all compounds, except for the limit of quantification (±20%). The present work describes the first application of microextraction by packed sorbent for the concentration of cocaine and metabolites extracted from hair samples. Graphical abstract.

A valid method to determine the site of drowning.

The diatom test is considered a useful aid in determining the site of death from drowning. Nevertheless, there is disagreement within the scientific community concerning its reliability, and its findings have been challenged and sometimes overturned in courts of law. Using a model based on animal experimentation, we have developed a diatom test to discriminate between the locations of drowning sites from different aquatic systems. We carried out a complementary combination of quantitative and qualitative analyses together with a statistical analysis based on the Kullback-Leibler distance of the samples. A restrictive selection of exclusive diatom species from each reservoir was also made. This approach allowed us to validate the usefulness of the diatom test in determining the location of the site of drowning.

The Application of Flow Cytometry as a Rapid and Sensitive Screening Method to Detect Contamination of Vitreous Humor Samples and Avoid Miscalculation of the Postmortem Interval.

Research into maximizing the speed, precision, and reliability of estimating the postmortem interval (PMI) has been a recurring object of investigation and methodologies based on the vitreous humor (VH) have provided good results. However, contamination from causes not readily apparent, such as blood, can occur, and thus lead not only to an erroneous estimation of PMI, but also interfere with the correct identification of other substances in the VH. We have developed a flow cytometry method which quantifies blood contamination and is able to detect erythrocytes in 1:750,000 dilution of contaminated VH which affects the results of hypoxanthine. It is an improvement on the previous more complex mass spectrometry method, being faster, more sensitive, and readily available. As such, it could be proposed for the rapid screening of appropriate samples by detecting and eliminating blood contaminated samples from PMI estimation.

Sex estimation from the tarsal bones in a Portuguese sample: a machine learning approach.

Sex estimation is extremely important in the analysis of human remains as many of the subsequent biological parameters are sex specific (e.g., age at death, stature, and ancestry). When dealing with incomplete or fragmented remains, metric analysis of the tarsal bones of the feet has proven valuable. In this study, the utility of 18 width, length, and height tarsal measurements were assessed for sex-related variation in a Portuguese sample. A total of 300 males and females from the Coimbra Identified Skeletal Collection were used to develop sex prediction models based on statistical and machine learning algorithm such as discriminant function analysis, logistic regression, classification trees, and artificial neural networks. All models were evaluated using 10-fold cross-validation and an independent test sample composed of 60 males and females from the Identified Skeletal Collection of the 21st Century. Results showed that tarsal bone sex-related variation can be easily captured with a high degree of repeatability. A simple tree-based multivariate algorithm involving measurements from the calcaneus, talus, first and third cuneiforms, and cuboid resulted in 88.3% correct sex estimation both on training and independent test sets. Traditional statistical classifiers such as the discriminant function analysis were outperformed by machine learning techniques. Results obtained show that machine learning algorithm are an important tool the forensic practitioners should consider when developing new standards for sex estimation.

High variation in hypoxanthine determination after analytical treatment of vitreous humor samples.

In the field of legal medicine the correct determination of the time of death is of great importance because an error in calculating the post-mortem interval (PMI) could be crucial in a criminal investigation. The quantification of hypoxanthine (Hx) concentration in the vitreous humor (VH) as a means to estimate PMI is useful when dealing with a recent time interval of death and has several advantages over other biological matrices. However, due to its viscous nature, VH has to be liquefied prior to its analysis, and the different procedures to liquefy it may cause alterations in Hx concentration, and hence in estimation of PMI. The aim of this study was to determine differences in Hx concentration after applying several pre-analytical treatments to different aliquots of the same VH sample. Enzymatic digestion, sonication, centrifugation and heat were the liquefying methods applied to reduce the viscosity of 105 VH samples. Analysis was performed using solid phase extraction and liquid chromatography in tandem with mass spectrometry. Statistically significant differences in the measured Hx concentrations were found between enzymatic digestion and the other three methods analyzed (sonication, centrifugation, and heat). Additionally, high data dispersion was found under heat treatment but was not observed for centrifugation or sonication, where similar and more moderate results were found. We conclude that high variations in Hx determination are observed when enzymatic digestion or heat treatment is used and we therefore recommend sonication or centrifugation to quantify Hx concentration in the VH.

Valoração do dano estético nos acórdãos do Tribunal de Justiça de Minas Gerais / Assessment of the aesthetic damage in the jurisprudence of the Court of Justice, Minas Gerais

Introdução: Atualmente no Brasil, o valor da indenização por dano extrapatrimonial (moral) tem sido fixado por arbitramento do juiz. O dano moral não é passível de mensuração econômica e, sendo assim, torna-se necessária a utilização de parâmetros para avaliação do dano. Objetivo: Analisar a frequência do dano estético nas jurisprudências do Tribunal de Justiça de Minas Gerais (TJMG) e identificar o dispositivo legal ou parâmetro que o magistrado empregou para fixar a indenização. Material e Método: Foi realizado levantamento das ações julgadas no TJMG nos acórdãos no período de 2009 a 2012, sendo o cirurgião-dentista processado pelos pacientes. Resultados: Em 76 processos observou-se que somente o método descritivo - análise abstrata - foi utilizado para expor a alteração estética. Em relação à frequência do dano estético nas jurisprudências, dos 35 processos em que houve absolvição dos cirurgiões-dentistas, 94,3% não solicitaram a reparação do dano estético como terceiro gênero. Em relação aos cirurgiões-dentistas condenados, em 63,4% dos 41 processos encontrados não existiu solicitação de dano estético. Em 17,1% dos casos houve solicitação de reparação ao dano estético, enquanto que em 19,5% a solicitação de dano estético foi de forma subentendida. Conclusão: A maioriados julgados analisados não considerou o dano estético como um terceiro gênero e não foi identificado um parâmetro objetivo pelos magistrados para fixar o valor da indenização.

Introduction: Currently, in Brazil, the value of compensation rate for off-balance impairments has been stipulated according to judge's conviction. However, these impairments are not measurable, making necessary the use of specific parameters for assessment and evaluation. Aims: To analyze the frequency of aesthetic damages in the Court of Justice of Minas Gerais. Additionally, to identify the parameters used by the judge in order to stipulate the value of compensation rate. Material and methods: Lawsuits of the Court of Justice of Minas Gerais, dating from 2009 to 2012, in which dentists were suited by patients, were selected for detailed analysis and systematic interpretation. Results: In 76 judgments, only the descriptive method was used to stress the aesthetics damage. In relation to the frequency of these impairments, considering the dentist's acquittal, 94.3%of the 35 judgments did not requested compensation rate for aesthetic damages. On the other hand, considering the dentist's conviction, the compensation rate for aesthetic damages was not requested in 63.4% of 41 judgments. In general, in 17.1% of the judgments the compensation rate for aesthetics impairments was requested, while into 19,5% of these judgments the request was subjective. Conclusion: Most of the analyzed judgmentsdid not consider the aesthetic damage as a third type of compensable impairment. In addition, an objective parameter for the stipulation of compensation rates was not detected into the judgments.

Cardiac inhibitory reflex as a cause/mechanism of death.

The role of cardiac inhibitory reflex as a potential cause of death is still a matter of debate. This study reports two cases of death under unusual circumstances. Case 1 corresponds to a man found hanging where the role of ligature compression of the carotid sinus became relevant as a possible explanation of death. In Case 2, the participation of a vasovagal syncope was clearly triggered by the laryngoscopic procedure. It is proposed that cardiac inhibitory reflex should be taken into account in those cases of unexpected death, which fulfills the following three criteria: (i) The investigation of the circumstances of the death is consistent with a hypothesis of cardiac arrest. (ii) A typical triggering peripheral stimulus is present. (iii) The performance of a complete autopsy cannot rule out the participation of a cardiac inhibitory reflex in the cause of death.

Applications of Tandem Mass Spectrometry (LC-MSMS) in estimating the post-mortem interval using the biochemistry of the vitreous humour.

It is widely accepted that the quantification of potassium concentrations ([K+]) and hypoxanthine ([Hx]) in the vitreous humour is useful in estimating the time of death within a recent time interval. Despite all the advances made in this area, it is well recognized that difficulties in calibration, validation and the use of different methodologies and instrumentation from different laboratories may lead to obtaining different concentrations from the same sample. The extraction of the vitreous humour itself should also be carried out with a precise technique, taking care during the procedure to avoid causing any vascular injury that might lead to haematic contamination. Any fluid that is not clear could lead to erroneous results and should be discarded. We present a new LC-MSMS method developed for quantitative and qualitative analysis of [Hx] (valid also for guanine and xanthine), and qualitative determination of uric acid in vitreous humour. We also introduce a methodology to assess haematic contamination in order to improve the estimation of time since death. The method was fully validated in terms of linearity, sensitivity, imprecision, analytical recovery, extraction and process efficiency and matrix effect.

PMICALC: an R code-based software for estimating post-mortem interval (PMI) compatible with Windows, Mac and Linux operating systems.

In legal medicine the correct determination of the time of death is of utmost importance. Recent advances in estimating post-mortem interval (PMI) have made use of vitreous humour chemistry in conjunction with Linear Regression, but the results are questionable. In this paper we present PMICALC, an R code-based freeware package which estimates PMI in cadavers of recent death by measuring the concentrations of potassium ([K+]), hypoxanthine ([Hx]) and urea ([U]) in the vitreous humor using two different regression models: Additive Models (AM) and Support Vector Machine (SVM), which offer more flexibility than the previously used Linear Regression. The results from both models are better than those published to date and can give numerical expression of PMI with confidence intervals and graphic support within 20 min. The program also takes into account the cause of death.

Predicting adult stature from metatarsal length in a Portuguese population.

Stature can be considered one of the "big four" parameters to be ascertained within the biological profile in cases of forensic anthropology. However, the most reliable available methods for stature estimation require the preservation of the long bones, but since this is very often not the case, the development of alternative methods, based on distinct bones, is mandatory. Therefore, in the present work the reliability of the first two metatarsal bones in reconstructing stature is tested. The data consist of length measurements taken from the first two metatarsals removed from documented cadavers of known stature. The sample for this study consists of 220 metatarsals, namely 110 first metatarsals and 110 second metatarsals collected during the autopsies carried out in the National Institute of Legal Medicine in Portugal. The aim was to propose regression equations for the Portuguese population and test the formulae proposed by other authors to determine adult stature using metatarsal bones. We found that when estimating stature from measurement of the metatarsals, the best correlation was that obtained from the relationship with the maximum length of the 2nd metatarsal. The corresponding regression equation is as follows: S=790.041+11.689M2.

Characterization of lesions in hanging deaths.

Hanging is a common method of committing suicide and a routine task in medico-legal autopsies. The hanging mark is the most relevant external sign and its characteristics are well known, but, for unknown reasons, there are major differences in reports on internal findings. We retrospectively studied 228 consecutive cases of hanging deaths. A complete standard autopsy was performed for every case. We investigated the association between the characteristics of the hanging mark and the frequency of bone, cartilage, soft tissue, and vascular injuries with the mode of suspension. Most cases (75.3%) presented some kind of bone or cartilage fracture, but these were unrelated to any of the variables studied. Vascular lesions are clearly more infrequent: intimal injuries were found in the carotid artery (9.1%), the jugular vein (2.2%), and ruptures of the carotid adventitial layer (21.7%). These could be partially associated with the use of a hard fixed noose and body weight.

Y-STR haplotype diversity in distinct linguistic groups from East Timor.

East Timor is a country which harbors multiple ethnolinguistic groups generally assigned to an Austronesian or Papuan ancestry. The present study aimed to characterize Y-chromosome haplotype diversity in East Timor, and to test possible population structures based on linguistic and/or geographical information. Using a set of 12 Y-chromosome-specific STRs (DYS19, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, and DYS439), haplotypes were established in 342 individuals from 12 linguistic groups (Tetum, Kwaimina, Galoli, Wetarese, Dawan, Mambai, Kemak, Tokodede, Bunak, Makasai, Makalero, and Fataluku) belonging to the three major ethnolinguistic groups in East Timor: two from the Timorese-Austronesian branch (Fabronic and Ramelaic), and a third including languages related to a Trans-New Guinea phylum (Papuan). High values of haplotype diversity, average gene diversity, and mean number of pairwise differences per locus were found in all 12 linguistic groups, except for the Wetarese from the island of Ataúro. Analysis of genetic variance (AMOVA) and pairwise genetic distance analysis showed that the East Timor population is genetically structured, and if the Bunak and Wetarese are excluded, samples group well with respect to their language affinities, and furthermore, the most genetically homogeneous groups are those following the broad ethnolinguistic classifications. Bunak and Wetarese behave as outsider groups, and are genetically more closely related to populations classified in a different linguistic group.

Allele frequencies and haplotypes of 8 Y-chromosomal STRs in the Santa Catarina population of southern Brazil.

Allele frequencies and haplotypes of eight Y-chromosomal short tandem repeats (STRs), DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392 and DYS393 were determined in a sample of 109 males from Santa Catarina. The origin of this southern Brazilian population is mainly from Portuguese people, namely from Azores archipelago.