A rapid and convenient LC/MS method for routine identification of methamphetamine/dimethylamphetamine and their metabolites in urine

A rapid and sensitive LC/MS method was developed for the simultaneous analysis of N,N-dimethylamphetamine (DMA), N,N-dimethylamphetamine N-oxide (DMANO), methylamphetamine (MA) and amphetamine (A) in urine samples. Employing an Alltech C18 column for solid phase extraction followed by LC/MS analysis using an Alltech Platinum EPS C18 column with a mixture of ammonium formate (0.01 M, pH 3) and acetonitrile (77:23, v/v) as mobile phase at a flow rate of 0.2 mL/min, simultaneous identification and quantitation of A, MA, DMA and DMANO in urine can be achieved using a 5-min chromatographic run. The calibration ranges were 0.10-3.0 micro g/mL for DMANO, 0.05-3.0 micro g/mL for DMA and 0.05-5.0 micro g/mL for both MA and A. The intra-, inter-day precision and accuracy for all analytes, spiked at three different concentrations in quality control samples, were in the ranges of 1.7-8.6, 4.1-10.0, -11.6 to 12.9%, respectively. The newly developed method was applied to the analysis of urine samples obtained from 118 suspected MA/DMA abusers, with the presence of MA confirmed in their urine samples under the drug-use surveillance program. Of these 118 samples, 43 were found to contain DMANO and 11 with both DMANO and DMA.     

An investigation of model forensic bone in soil environments studied using infrared spectroscopy

Infrared spectroscopy has been used to examine changes to bone chemistry as a result of soil burial. Pig carcasses were buried as part of a controlled field study, and pig bone was used in soil environments established in the laboratory. The variables of species type, bone pretreatment, soil type and pH, moisture content, temperature, and burial time were investigated. The crystallinity index (CI) and the organic and carbonate contents of the bones were monitored. The data revealed decreasing trends in the organic and carbonate contents and an increase in the CI of the bone with burial time. An acidic soil environment and soil type are the factors that have the most influence on bone chemistry as a result of burial. The study demonstrates the potential of infrared spectroscopy as a straightforward method of monitoring the changes associated with aging of bones in a variety of soil environments.     

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.     

Employing glass refractive index measurement (GRIM) in fiber analysis: a simple method for evaluating the crystallinity of acrylics

The refractive index (RI) of 40 colorless acrylic fiber samples was determined by the immersion oil and Mettler hot stage method, implemented in the glass refractive index measurement (GRIM) instrument by Foster and Freeman. Low standard deviations for nearly all the samples were obtained and differentiation of fibers even with the same trade name was possible just on the basis of RI. Wide angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC) were employed to establish a linear correlation between RI and the degree of crystallinity. Insights on the structural order of this group of fibers is then obtainable by a quick and easy technique, adding value to casework comparisons.     

Evaluation of the rapid analyte measurement platform (RAMP) for the detection of Bacillus anthracis at a crime scene

The aim of this study was to evaluate the accuracy and reliability of the rapid analyte measurement platform (RAMP) for presumptive identification of Bacillus anthracis spores. Test samples consisted of serial dilutions of spore preparations of several Bacillus species, including B. anthracis, which were tested, using the RAMP Anthrax test cartridge, according to the manufacturer's instructions. The fluorescence labelled antibody-antigen complexes were detected in the portable reader after 15 min following sample addition. Dilutions of common environmental and household powders were also tested to identify possible false positive results. B. anthracis spores were identified reliably in test samples containing more than 6000 spores. The test kits were highly specific, showing no cross reactivity with other Bacillus species or any environmental powders tested. The RAMP system for detection of B. anthracis spores, from environmental samples, showed consistent results under a variety of analytical conditions, enabling the trained user to provide a rapid, accurate preliminary risk assessment of a suspected bioterrorism incident.     

The relationship between alcohol elimination rate and increasing blood alcohol concentration--calculated from two consecutive blood specimens

In the period 1991-2005, a blood-alcohol concentration (BAC) analysis was carried out at the Institute of forensic medicine in Novi Sad including 2023 two consecutive blood specimens using the Headspace Gas Chromatography method. Cases with no alcohol concentration values, as well as cases where blood samples were taken within 1 h after the criminal act, were not taken into consideration. Following this rule, 1198 cases were considered in this study and all samples were grouped in 29 ranges of BAC1 of delta(BAC) = 0.1 g/kg, starting from 0.1-0.19 g/kg to 2.9-2.99 g/kg of absolute alcohol. Gathered results and elimination curve differ from the zero-order model of elimination proposed by Widmark and point to an elimination process similar to a well-known Michaelis-Menten elimination kinetics model and its variants. Results reported in this study show dependence of alcohol elimination rate (beta-slope) and BAC value. The analysis of beta60-slope versus BAC shows that a correlation between beta60 (y) and BAC (x) has a logarithmic trend line. The value of alcohol elimination rate shows a slight increment with increase of BAC alcohol, with the mean value of beta60 = 0.221 +/- 0.075 g/kg. Differences in values of beta60 among consecutive intervals of delta(BAC) = 0.1 g/kg are not significant (p>0.05). When obtained samples were grouped into ranges of 0.5 g/kg each in these intervals beta60 had the following values by range: 0.1-0.49 g/kg = 0.139 g/kg +/- 0.035; 0.5-0.99 g/kg = 0.184 g/kg +/- 0.043; 1-1.49 g/kg = 0.213 g/kg +/- 0.052; 1.5-1.99 g/kg = 0.239 g/kg +/- 0.058; 2-2.49 g/kg = 0.265 g/kg +/- 0.073; 2.5-2.99 g/kg = 0.306 g/kg +/- 0.096. Differences in values of beta slope among consecutive intervals of delta(BAC) = 0.5 g/kg are significant (p<0.01). The elimination curve in the BAC interval 0.5-2.5 g/kg has a linear trend, while beta-slope (y)/BAC (x) correlation is given as beta60 = 0.15 g/kg + (0.05 g/kg x BAC). Retrograde calculation of the blood alcohol concentration in tempore criminis (BAC(tc)) based on the determined alcohol concentration in the blood specimen (BAC(t)) shows a statistically significant difference between BAC(tc) calculated using a standard zero-order model versus corrected methodology. The higher the BAC(t) and the longer the calculation time, the greater and statistically more significant (p<0.01) is the difference between the calculated values of BAC(tc).     

Dominance of pre-analytical over analytical variation for measurement of methadone and its main metabolite in postmortem femoral blood

On the basis of simultaneously sampled postmortem blood specimens from the left and right femoral veins the pre-analytical variation of methadone measurements was evaluated and compared to the analytical variation. The material consisted of a series of 27 duplicate samples from routine autopsy cases comprising mainly drug addicts. A chiral LC-MS/MS method was used for measurement of the R- and S-enantiomers of methadone and its main metabolite 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP). The analytical CV% was determined to be in the range 3-4% for methadone enantiomers and 4-6% for EDDP enantiomers. The total measurement uncertainty (CV(T)) was estimated from the pre-analytical variation (CV(PA)), analytical variation proper (CV(A)), and variation related to calibration (traceability) (CV(Cal)) according to the relationship CV(T) = [CV(2)(PA) + CV(2)(A) + CV(2)(cal)](0.5). Uncertainty related to calibration concerned a component related to the purity of drug reference compound and a contribution from the production of calibrator solutions (CV(Cal)<1%). Pre-analytical sampling variation was estimated from the duplicate measurements of blood samples after subtraction of the analytical component. The pre-analytical variation amounted to a CV% of 19-21% for R- and S-methadone and 30-38% for R- and S-EDDP, i.e. considerably larger than the other components. Due to the squared addition principle, the resulting total uncertainty (CV(T)) became largely identical to the CV(PA), i.e. 19-21% for R- and S-methadone and 31-38% for R- and S-EDDP enantiomers. Accordingly, CV(T) exceeded CV(A) by a factor 5 or more. Dominance of the pre-analytical component of variation may also be likely for other compounds measured in postmortem blood samples. Thus, the width of the 95%-uncertainty interval (+/-2CV(T)) for a postmortem measurement is largely determined by the pre-analytical component of variation. This should be kept in mind when judging on the uncertainty of postmortem measurement results.     

PRAXIS--combined mu-Raman and mu-XRF spectrometers in the examination of forensic samples

A major role of forensic entomology is to estimate the post-mortem interval. An entomologist's estimate of post-mortem interval is based on a series of generally valid assumptions, error in any of which can alter the accuracy of an estimate. The initial process of collecting and preserving maggots can itself lead to error, as can the method of killing and preservation. Since circumstances exist where it is not possible to rear maggots, methods of killing and preservation can be vital to preserving the integrity of entomological evidence. In this study, a number of preservation techniques used at crime scenes and in mortuaries were examined, and their effect on feeding third-instar larvae of Calliphora augur and Lucilia cuprina evaluated. The preservatives used were 70, 75, 80, 90 and 100% EtOH, Kahle's solution and 10% formalin. Each treatment was replicated three times. The effect of handling on first- and second-instar, feeding and post-feeding third-instar larvae of C. augur was also examined and compared to unhandled controls. Finally, the effects of preservatives were noted when larvae of C. augur and L. cuprina were placed into preservatives alive. It was found that continued handling is detrimental to specimens because preservative evaporates from both the vial and the specimens. No single preservative type was found to be entirely suitable for both species if DNA retrieval is desired. Specimens placed into most preservatives alive exhibited adverse colour changes, desiccation, sunkeness and agglomeration. It is concluded that the reaction to preservative type might be species specific and that different instars of the same species might also react differently.     

不动产适用善意取得制度的限度

不动产登记簿的公信力和善意取得制度是两种构造迥异的物之交易信赖保护机制。善意取得制度以占有不足以充分表征动产所有权为构造前提,以竭力衡量所有权人与善意第三人的利益关系为轴心;不动产登记簿的公信力制度以不动产登记簿可以作为权利外观为构造前提,以完备的不动产登记制度为根基。善意取得制度的效果只能是第三人由无权利人取得物权;不动产登记簿的公信力所具有的效果不但有积极信赖保护与消极信赖保护之分,且其积极信赖保护的内容除由无权利人取得物权外,还包括由有权利人取得物权、受领给付、获得权利顺位等。因此,以善意取得制度保护不动产交易的便捷与安全,其局限非常明显。物权法第106条应限缩解释为主要适用于动产,不动产交易的信赖保护可通过解释物权法第16条来实现。     

Genetic citizenship: DNA testing and the Israeli Law of Return

The Israeli State recently announced that it may begin to use genetic tests to determine whether potential immigrants are Jewish or not. This development would demand a rethinking of Israeli law on the issue of the definition of Jewishness. In this article, we discuss the historical and legal context of secular and religious definitions of Jewishness and rights to immigration in the State of Israel. We give a brief overview of different ways in which genes have been regarded as Jewish, and we discuss the relationship between this new use of genetics and the society with which it is co-produced. In conclusion, we raise several questions about future potential impacts of Jewish genetics on Israeli law and society.     

Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.     

Bone Mineral Change During Experimental Calcination: An X‐ray Diffraction Study

The effects of calcination (400–1200°C) on pig bones have been studied using powder X‐ray diffraction (XRD) and secondary modifications, such as color change and weight loss. The characterisation by powder XRD confirmed the presence of the crystalline phase of hydroxyapatite, and comparison of the results obtained at different temperatures suggested that at 650°C, all the organic components and carbonate substitutions were completely removed. Accordingly, these samples were white. In addition, the crystallinity degree and the crystallite size progressively increased with the calcination temperature until 650°C, remaining stable until 1200°C. Below 650°C, bone samples presented organic compounds, resulting in background noise in the diffractogram and gray or black color. In addition, impurities in the lattice correspond to low crystallite sizes.     

Forward,Gillick: Are competent children autonomous medical decision makers? New developments in Australia

Another chapter has opened in the tortured history of the status of Gillick competence. Never before has Gillick been extended to permit a mature child to make autonomous medical decisions over and above the curial ‘parens patriae’ power. In 2013, two judicial decisions promulgated from different Australian courts are in conflict over this most fundamental of questions. This Article situates the law of the ‘parens patriae’ power in Australia and, drawing on overseas conceptualizations of analogous doctrine, explores the bases for and potential consequences of this conflict.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

Determination of DNA yield rates in six different skeletal elements in ancient bones

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18^th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.     

PrepFiler Express BTATM Lysis Buffer Combined with Silicon Microbeads for Rapid DNA Extraction from BoneCSCD

Objective: To establish a convenient and rapid method for extracting DNA from bone. Methods: Fifteen long bone samples were washed and sterilized. The skeletal fragments were obtained by electric drill, and lysed by PrepFiler Express BTATM lysis buffer. DNA was then manually extracted by silicon microbeads for further analysis. Results: STR genotyping was successfully obtained in 14 out of the 15 samples, and the detection rate was 93.33%. Conclusion: The method for DNA extraction from bone established in present study is convenient, quick, effective, and with a strong applicability, which is worth spreading and applying. © 2017 by the Editorial Department of Journal of Forensic Medicine.     

Chemical Differentiation of Osseous and Nonosseous Materials Using Scanning Electron Microscopy–Energy-Dispersive X-Ray Spectrometry and Multivariate Statistical Analysis

Identification of osseous materials is generally established on gross anatomical features. However, highly fragmented or taphonomically altered materials may be problematic and may require chemical analysis. This research was designed to assess the use of scanning electron microscopy–energy-dispersive X-ray spectrometry (SEM/EDX), elemental analysis, and multivariate statistical analysis (principal component analysis) for discrimination of osseous and nonosseous materials of similar chemical composition. Sixty samples consisting of osseous (human and nonhuman bone and dental) and non-osseous samples were assessed. After outliers were removed a high overall correct classification of 97.97% was achieved, with 99.86% correct classification for osseous materials. In addition, a blind study was conducted using 20 samples to assess the applicability for using this method to classify unknown materials. All of the blind study samples were correctly classified resulting in 100% correct classification, further demonstrating the efficiency of SEM/EDX and statistical analysis for differentiation of osseous and nonosseous materials.     

Characteristics and Frequency of Chipping Effects in Near‐Contact Gunshot Wounds

The presence of “chipping” or “flaking” around the edges of gunshot entry wounds has been described among the characteristics of gunshot wounds in bone. In this study, the real frequency of such a peculiar feature was investigated. The presence of “chipping” was assessed on 22 gunshot wounds fired at a near‐contact range on bovine ribs with 9‐mm bullets. As controls, five samples were shot with a 3 cm range, and five from 40 cm. In 77% of cases shot at near‐contact range, a detachment of small fragments of the upper layers of bone was detected, mainly with a circumferential disposition, whereas this feature was lacking in control samples. The study demonstrated the frequency of “chipping” and that it may probably be due to a combined ballistic effect of impact of the bullet itself and expansion of gases. It may be thus considered indicative of close‐range shots.     

Sexing North American Eskimo and Indian innominate bones with the acetabulum/pubis index

The acetabulum/pubis index was tested on American Eskimo and Indian samples of innominate bones to which sex had been assigned by visual inspection. The index alone sexed 91 and 89% of the Eskimo and Indian samples, respectively. Subsequent sorting of misclassified specimens with femur head diameter resulted in 96 and 99% rates of classification. The index and a measurement of ischial height were used in a discriminant function model which correctly identified the sex of 93% of the Eskimo and 92% of the Indian bones. Using femur head diameter to sort those specimens which were misclassified by their discriminant function score yielded classification rates of 96 and 98%. These results are compared to a range of 92 to 100% obtained with the index in earlier studies of American black and white pelvic bone samples.