Morphological study of human hair infected with Microsporum canis

Some aspects in forensic medical examination of hairs damaged by Microsporum canis are discussed. The data of microscopic, fluorescent, and serological studies of damaged hairs and methods for their examination are presented.     

A method for the longitudinal sectioning of single hair samples

A simple technique is presented for the longitudinal sectioning of hair samples without the need for any embedding medium. The technique applies to single hair samples that are placed in a groove of a particular depth and can either be scraped or cut to the desired level. Planar sections are obtained that reveal the internal structure of the sample and are ideal for the application of surface analytical techniques for the study of internal transverse and longitudinal distributions of xenobiotics.     

Determination of hair group by preserved hair cell bulbs

Difficulties appearing in determining hair group were the reason for conducting this research for the purpose of finding additional possibilities to solve the mentioned problem. It was established that the preliminary cell coloring, related with determination of hair sex, does not influence a consequent detection of antigens. Methods or the fixation of material were selected. The most suitable reagents and their titers as well as different time periods of absorption for detecting antigens A and B are offered. All stages of examination are described in detail.     

A simplified method for mitochondrial DNA extraction from head hair shafts

DNA isolation from hair shafts can involve a number of steps, each of which adds time to the procedure and increases the risk of contamination. A simple alkaline digestion procedure that directly dissolves hairs was developed and compared with a widely used glass grinding/organic extraction method, using samples collected from 30 volunteers with varying population ancestries, hair colors, and hair treatments. A 203 bp mtDNA product could be amplified from 90% of samples extracted by alkaline digestion and 73% of hairs extracted by glass grinding. DNA obtained from alkaline digested hair generated equal or greater amplification success for virtually all criteria examined, and mtDNA sequences matched buccal control sequences in all cases. The two methods were similar in DNA yield (amplification success at template dilution) and quality of DNA obtained (amplicon length). Alkaline digestion of hair shafts required 6-7 h to complete, compared to 22-24 h for glass grinding, and proved a less laborious yet equally robust method for mtDNA extraction.     

Immunochemical studies on blood group A substance from human hair

Blood group A-active substance was extracted from urea-treated human hair uith methanol-ethyl ether 1:1, v/v) or chloroform-methanol (1:1, v/v). The serological activity of blood group A substance in the hair was destroyed by A-decomposing enzyme from Clostridium tertium with concomitant development of blood group H activity. It is concluded therefore that the extract from the hair of group A contained blood group A-active glycolipid with N-acetylgalactosamine as the non-reducing sugar.     

Determination of ABO blood group markers in single hair segments

Determination of ABO-blood group on human hair segments of 79 individuals was performed by means of modified Yada absorption-elutions technique. The studies were carried out with 4 micron thin cross sections of hair. In 79% of the cases it was possible to establish the correct blood group. Individual hair segments of 0.5 cm length yielded reliable results. Antigen identification was best with blood group B and most difficult of the AB type.     

A new 39-plex analysis method for SNPs including 15 blood group loci

A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1-1.1) x 10(-14), and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen-M?ller's W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22 x 10(-17). These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.     

A novel method for the identification of saliva by detecting oral streptococci using PCR

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.     

An ELISA method for the identification of salivary amylase

An ELISA method for the detection of salivary amylase in dried stains using a monoclonal anti-human salivary amylase antibody was developed. Studies demonstrated the assay to be sensitive down to 0.0002 Sigma units and showed a linear response between absorbance and salivary amylase activity between 0.002 and 0.2 units. The assay showed no cross reactivity with either commercially purchased human pancreatic or bacterial amylase. Sample studies utilizing swabs from several human body fluids showed that 100% of all saliva containing swabs (sixteen of sixteen) and 13% of non-saliva human body fluid swabs (eight of sixty-three) showed a net absorbance with the method. Of these eight non-saliva swabs yielding a net absorbance, none exceeded a salivary amylase activity of 0.003 units. In contrast, only three of the sixteen saliva-containing swabs (swabs produced from saliva diluted 1:5, 1:6, and 1:10, respectively) showed an activity below 0.2 units. Of these swabs, the 1:100 dilution showed the lowest activity (0.048). This value is still more than ten times that of the non-saliva containing swab with the highest activity.     

A silica-based mitochondrial DNA extraction method applied to forensic hair shafts and teeth

The purpose of this study is to evaluate the applicability of a nonorganic DNA extraction method for use in the analysis of environmentally compromised forensic hair shaft and tooth samples. The condition of the samples included cases of water decomposition, severe incineration, and varying stages of putrefaction. Enzymatic amplification and manual sequencing of the first segment of the mitochondrial hypervariable region were performed successfully on each of the 20 autopsied individuals. The results indicate that the silica-based extraction method produces mtDNA suitable for genetic identification from forensic samples including hair shafts and teeth.     

A new method for collection and identification of gunshot residues from the hands of shooters

This work presents a novel collection method for gunshot residues (GSR) using a sampling procedure based on ethylenediaminetetraacetic acid (EDTA) solution as a complexing agent on moistened swabs. Detection was via a sector-field inductively coupled plasma mass spectrometry (HRICP-MS). The proposed collection and analytical method allowed detection of antimony (Sb), barium (Ba) and lead (Pb) after .38 shot tests. at detection limits of less than 1 microg L(-1) in four different areas of the hands of volunteers. This paper includes a discussion concerning hand areas near the thumb and forefinger as being more suitable for GSR collection as well as a comparison between differences observed using 2% diluted EDTA. 2% nitric acid solution, and simple deionized water as collecting solutions, proving the superior efficiency of EDTA in GSR recoveries.     

Shadow positioning technique: a method for postmortem identification

Radiology is increasingly being used as a means of postmortem identification. We have devised a shadow positioning technique by which a postmortem radiograph of a skeletal part can exactly duplicate an antemortem radiograph, thus, faciliating identification by comparison of the antemortem and postmortem radiographs. The antemortem radiograph can be of any skeletal part and taken in any position.     

测定单根毛发中吗啡含量的放射免疫方法

目的 建立测定单根毛发中吗啡含量的放免方法。方法 用卵清蛋白-琥珀酰吗啡作免疫原,免疫新西兰白兔获得高品质抗血清;HPLC纯化~(125)Ⅰ-吗啡,建立放射免疫方法,测定正常人和吸毒人员单根毛发。结果 抗体亲和常数为3.25×10~(11)L/M,放化纯度为95％,比放射性112μCi/μg;方法的灵敏度为0.01ng/ml。对5例正常人及5例戒毒所吸毒人员的单根毛发进行了检测。单根毛发长度9～24cm,重量为0.7～2.1mg,5例正常人测值为1.75±0.37ng/mg(x±s);5例吸毒人员测值为471±204ng/mg(x±s)。结论 所建方法可准确定量单根毛发中吗啡的含量。     

Lifestyle illicit drug seizures: A routine ESI-LC-MS method for the identification of sildenafil and vardenafil

For street samples suspected of containing the phosphodiesterase-5 inhibitors sildenafil (Viagra(?)) and/or vardenafil (Levitra(?)), including powders or adulterated herbal supplements, a chemical analysis is needed to provide confirmatory identification of these illegally procured substances. Sildenafil and vardenafil are structurally similar and it is difficult to differentiate between them, as previous mass spectrometric studies have shown the two drugs to produce similar fragmentation patterns. The use of tandem mass spectrometry can produce confirmatory data, but the technique requires a high level of technical expertise. We have developed an electrospray ionization-liquid chromatography-mass spectrometry (ESI-LC-MS) method that allows differentiation between these two structurally similar molecules via in-source fragmentation in combination with an ion trap mass spectrometer. A very stable gas phase ion is formed during in-source fragmentation of vardenafil; the combination of the stability of this ion and the longer residence time for the ion in the ion trap results in a very strong signal. This feature results in a method that can provide clear differentiation between sildenafil and vardenafil while at the same time requiring less expertise from the routine analyst to confirm the presence or absence of the two compounds.     

Use of the isoelectric focusing method in examining hair proteins

Method of isoelectric focusing in thin and ultrathin layer of polyacrylamide gel followed by staining the polypeptide fractions with argentum was used to investigate human hair keratins. Method makes it possible to determine the polymorphism of keratins in hair even 1,5-2 cm long. It helps to obtain the additional information in medicolegal expertise of hair affinity.     

Last performance with VIAGRA: post-mortem identification of sildenafil and its metabolites in biological specimens including hair sample

A 43-year-old man was found dead in a hotel room during a sexual relation with a colleague.He was treated both for cardiovascular disease and for erectile dysfunction with VIAGRA. A pillbox was found in the room with several tablets of verapamil (Isoptine), trimetazidine (Vastarel), yohimbine and bromazepam (Lexomil). A box of VIAGRA 25mg was found in his raincoat and two tablets were missing. His wife declared during the investigation that he was also treated by trinitrine. Autopsy revealed severe coronary artery sclerosis as well as signs of previous myocardial infarctions. Blood, urine, bile, gastric content and hair and representative tissues for histology were collected for toxicological analysis.Sildenafil and yohimbine were screened with liquid chromatography/mass spectrometry (LC/MS) and trinitrine with headspace injection (HS)/GC/MS. Verapamil and trimetazidine were identified and quantified with LC/diode array detection (DAD).Sildenafil was identified in blood, urine, bile and gastric content at 105, 246, 1206 and 754ng/ml, respectively. Hair concentration was 177pg/mg. The desmethyl metabolite was quantified in urine at 143ng/ml. Blood concentrations of verapamil and trimetazidine were measured at 659 and 2133ng/ml, respectively and were above therapeutic ranges. Trinitrine and yohimbine were not identified.These results confirm the absorption of sildenafil, verapamil and trimetazidine before the death and hair analysis indicates the chronic use of sildenafil.To the author's knowledge, this is the first report of a fatal sildenafil-verapamil association, probably by hypotension and cardiac dysrhythmia.     

A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin

The population of the Asian elephant is being dramatically reduced due to poaching of the ivory from the male. As poaching occurs in remote forests, it often takes weeks or longer for it to be discovered and it is therefore often very difficult to determine the sex of the decomposed body. Data suggest that in the recent past, over 2000 male elephants have been poached in South India. We have developed a technique based on molecular markers to determine that the carcass is an elephant and that it is a male. Using DNA sequence information from Genbank, we have developed two primer pairs: one for the mitochondrial DNA (mtDNA) and the other for the sex-determining region of Y chromosome (SRY) gene of the Indian elephant. After PCR amplification of known elephant DNA, we found that the mtDNA was common in both males and females, whereas the SRY-specific amplicon was observed only in the male.     

IBD法在堂表亲缘关系鉴定中的应用

目的 使用血缘一致性(identity by descent,IBD)法计算堂表亲缘关系的堂表关系指数(first cousin index,FCI)和累积堂表关系指数(combined first cousin index,CFCI),为IBD法鉴定两个个体是否具有堂表亲缘关系提供科学手段.方法 取124对堂表兄弟姐...     

度冷丁在豚鼠毛发中的分布研究

目的探讨度冷丁在毛发中的分布状况。方法通过动物实验-给豚鼠持续腹腔注射度冷丁,探讨度冷丁在豚鼠毛发中出现与消失的时间过程及注射剂量与毛发中的含量关系。结果给豚鼠隔日连续腹腔注射度冷丁7次,第4天可在豚鼠毛发中检出度冷丁,第8天达浓度高峰,停止注射后第5天检不出度冷丁;豚鼠毛发中度冷丁含量与度冷丁的注射剂量有关,但无明显的线性关系。结论研究结果为实际案例分析结果的评价提供依据。