Detection of benzodiazepines in different tissues, including bone, using a quantitative ELISA assay.

Benzodiazepines were analyzed in different tissue samples, including hone, by ELISA. The sensitivity of detection for different benzodiazepines was consistent with the manufacturer's reports of the cross reactivities of the antibodies used, with the greatest sensitivity for midazolam and the least for diazepam; in addition the pharmacokinetics was consistent with the known duration of action of the different benzodiazepines, with midazolam cleared rapidly, and diazepam slowly. Following intramuscular injection of 300 microg of midazolam at 16 h intervals for ten days, the drug was detectable in bone tissue samples obtained from skeletonized remains buried in soil at room temperature for three weeks.     

Identification and quantitation by 1H-NMR of metabolites in animal organs and tissues. An application of NMR spectroscopy in forensic science

1H-nuclear magnetic resonance (NMR) was applied to the study of metabolites in rat organs and tissues. From 1H-NMR spectra of D2O extracts of various organs and tissues, lactate, pyruvate, and some other substances were simultaneously identified and quantitated. On the basis of the results, the use of 1H-NMR for estimating post-mortem time was suggested.     

Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins

Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), alpha 2-macroglobulin (alpha 2-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human alpha 2-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next.     

Identification and quantification of phenobarbital in a mummified body 10 years after death

Abstract: This article reports the determination of phenobarbital in the mummified body of a 56‐year‐old man found completely mummified 10 years after his death. When alive, he was being treated for epilepsy with phenobarbital, and the recent analyses, performed with both immunochemical techniques and gas chromatography with mass spectrometry (GC‐MS), have revealed the presence of this substance in various tissues: the mean content of barbiturate in the mummified liver tissue was 93 μg/g, 216 μg/g in the heart, 17 μg/g in the lungs, 12 μg/g in muscles, and 31 μg/g in the skin. Preliminary screening tests with immunochemical techniques to evaluate the presence of other drugs were also performed. The sample resulted negative for all substances tested. Phenobarbital can be identified and quantified thanks to its excellent chemical stability and a hypothesis of what the concentrations in the fresh tissue could have been has also been reported.     

Utility of immunoassay in drug screening in skeletal tissues: sampling considerations in detection of ketamine exposure in femoral bone and bone marrow following acute administration using ELISA

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.     

Identification and quantification of p-hydroxyethylamphetamine as a novel metabolite of ethylamphetamine in rat by gas chromatography-mass spectrometry

Ethylamphetamine and two major metabolites in urine, obtained after intraperitoneal administration of the drug to rats, have been isolated by gas chromatography-mass spectrometry. We have also synthesized an authentic sample of p-hydroxy-N-ethylamphetamine by a simple method. In rat, the metabolic pathway of ethylamphetamine is similar to that of amphetamine and methylamphetamine according to quantitative analysis of the major metabolites using a selected ion monitoring (SIM) method.     

Sexual dimorphism in the subadult mandible: quantification using geometric morphometrics

There have been numerous attempts, with varying degrees of success, to differentiate males from females on the basis of the immature skeleton. We investigate here whether the mandible can discriminate immature individuals by sex; the techniques we apply are from the field of geometric morphometrics. The application of these methods in forensic anthropology is still relatively new; thus, an important aspect of this research is that it demonstrates potential applications in this discipline. The sample comprises 96 known age and sex subadult individuals; the three-dimensional coordinates of 38 landmarks are analyzed using the shape analysis software morphologika. Multivariate regressions indicated no significant sexual dimorphism in the subadult sample; this result is supported by poor cross-validated classification accuracy (59%). Our results suggest that the subadult mandible is not dimorphic (to the extent that dimorphism is not evident within the sample we studied); thus, sex determination using previously described criteria is likely to yield poor results.     

The quantification of fingerprint quality using a relative contrast index

Research into fingermark enhancement techniques has traditionally used visual comparisons and qualitative methods to assess their effectiveness based on the quality of the developed fingermark. However, with increasing research into the optimisation of these techniques the need for a quantitative evaluative method has arisen. Parameters for acceptable fingerprint quality are not well defined and generally encompass clear, sharp edges and high levels of contrast between the fingermark ridges and background material. Using these current parameters, a conclusive measurement of fingerprint quality and thus the effectiveness of development techniques cannot be achieved.This study presents a model through which an aspect of fingerprint quality can be objectively and impartially measured based on a relative contrast index, constructed through measuring the reflective intensity of the fingermark ridges against the background material. Using a fibre-optic spectrophotometer attached to a microscope with axial illumination, the intensity counts of the ridge detail and background material were measured and a logarithmic contrast index constructed. The microscope and spectrophotometer parameters were experimentally tested using a standard colour resolution chart with known reflective properties. The protocol was successfully applied to four sample groups: black inked fingerprints on white paper; latent fingermarks on white paper developed separately with ninhydrin and physical developer; and fingermarks in blood deposited on white tiles and enhanced with amido black. The contrast indices obtained quantitatively reflect the level of contrast and provide an indication of fingerprint quality through a numerical representation rather than previous qualitative methods. It has been suggested that the proposed method of fingerprint quantification may be viable for application in the forensic research arena as it allows the definitive measurement of contrast to aid the evaluation of fingermark detection and enhancement techniques.     

Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.     

Identification of animal species by protein radioimmunoassay of bone fragments and bloodstained stone tools

In forensics and archaeology, it is important to distinguish human from animal remains and to identify animal species from fragmentary bones and bloodstains. We report blind tests in which a protein radioimmunoassay (pRIA) was used to identify the species of six bone fragments lacking morphological specificity and 43 bloodstained lithic tools, knapped experimentally and soaked in blood of known animal and human origin. The submitters of the bone fragments and the bloodstained tools each listed a number of possible species, from which the testers selected the best match with the pRIA results. All six bone fragments were correctly identified: three humans, a deer, a dog, and a cow. Forty-three tools were stained with blood from a wide variety of species including ungulates, carnivores, a fish, and a bird. On 40 of these 43, at least one species (or blood-free control) was identified correctly. Some of the tools were stained with blood of two different species. A mixture of sheep and musk ox blood was correctly identified; in several other mixtures, only a single species was detected. Two tools with human blood and one with human sweat were correctly reported as human. There was a single false positive (one of three controls reported as weakly bovine) and no false negatives. We conclude that the pRIA technique shows a high degree of accuracy in discriminating human from animal bone fragments and bloodstains and in identifying animal species.     

基于psbA-trnH条形码的毒品原植物大麻及其混伪品的鉴别

目的 利用叶绿体上的间隔区psbA-trnH条形码序列鉴别毒品原植物大麻及其混伪品,为毒品原植物大麻的鉴定提供新方法.方法 采用PCR法扩增psbA-trnH间隔序列,双向测序后运用CodonCode Aligner、MEGA5.1软件进行数据处理,构建系统聚类树(NJ树).结果 psbA-trnH条形码序列分析表明大麻种内与种间遗传距离具有较大差异,基于psbA-trnH条形码构建的NJ树可鉴别大麻及其混伪品.结论 psbA-trnH 序列可以作为鉴定毒品原植物大麻及其混伪品的候选条形码序列,为毒品原植物大麻的快速、准确鉴定提供了新方法;DNA条形码技术在犯罪案件中涉及的植物鉴定中具有极大的应用前景.     

An ELISA using avidin-biotin complex for the determination of ABH group from saliva

The ABH group in a trace amount of saliva could be determined by an enzyme-linked immunosorbent assay (ELISA) using an avidin-biotin-peroxidase complex (ABC) technique. In this method ABH blood group substances as a solid phase are adsorbed to wells of a microtiter plate made of polystyrene. The primary antibody corresponding to the blood antigen adheres onto the wells, and reacts with the biotinylated secondary antibody. The previously formed ABC reagent is then added to the above wells, and finally the absorbance produced by the interaction of the peroxidase activity with a chromogenic substance is measured at 492 nm. This method proved to be clearly more sensitive for the detection of ABH blood groups in secretor-saliva than the conventional hemagglutination inhibition test. Also the ABH group of non-secretor-saliva could be easily determined by this method.     

Differentiation between human and chimpanzee in bloodstains by enzyme-linked immunosorbent assay (ELISA) using antihuman serum

An enzyme-linked immunosorbent assay (ELISA) for species identification of human bloodstains using two commercially available antisera against human serum is described. Human bloodstains were to be distinguished from those of chimpanzees and other animals using raw antisera, and the differentiation between human and chimpanzee became more evident when those antisera were absorbed with a small amount of chimpanzee plasma. Human bloodstains could clearly be identified by the present method even after 4 weeks of aging at room temperature.     

mtDNA 12S rRNA基因序列测定在腐烂动物肌肉样本鉴定中的应用

目的利用mtDNA12SrRNA基因序列测定法对宁波森林公安局查获的一例腐烂动物肌肉样本进行种属鉴定,并探讨该方法在腐烂动物肌肉样本鉴定中的应用价值。方法用酚/氯仿法从腐烂动物肌肉样本中提取出基因组总DNA,再用一通用引物通过PCR技术扩增mtDNA上12SrRNA基因的部分片段并进行序列测定。测序结果在GenBank上进行BLAST搜索,再利用DNAMAN软件进行同源性分析。结果扩增产物序列与东北虎的线粒体DNA的12SrRNA基因序列的部分片段的同源性高达99.9%。结论该动物样本为东北虎,本研究所用方法在野生动物案件的样本鉴定中有极高的应用价值。     

An examination of a contaminated seminal stain using absorption-elution and enzyme-linked immunosorbent assay (ELISA)

A semen stain, apparently contaminated with a detergent cleanser, was received for examination. The contamination interfered with the normal biochemical reactions of such stains. Treatment of the sample enabled ABO groups to be determined.     

Identification of blood stains and fetal tissue using genetic fingerprinting

Two cases from practical forensic serological investigations which could not be clarified by means of conventional serological methods, but could be resolved on the basis of the DNA finger print technique with oligonucleotide probes are presented. Both the comparison of the identity of the blood sample which had been stored for a long time and the determination of paternity in fetal material in a case of incest demonstrate the enormous possibilities of this method. However, some problems which still have not been resolved at least at present with regard to the evaluation and appraisal of the band patterns are also discussed.