The advantages of establishing the species classification of blood and other biological objects by the IFR in a quantitative modification

A quantitative modification of immunofluorescent test (IFT) is described. It was used for species identification of blood stains on material evidence objects. This modification is 1000 times more sensitive than electrophoretic methods traditionally used for this purpose in forensic medicine. Computer processing of the results helps objectively and persuasively determine the species appurtenance of blood and other biological objects. Results of the test represented as histograms should be attached to forensic medical conclusions as proofs for the court.     

Detection of semen in stains on material evidences by quantitative immunofluorescence test

Russian and foreign methods used in forensic medicine for detection of the semen in stains on material evidences are compared. The potentialities of quantitative immunofluorescence test for detection of the semen in stains on material evidences, developed at Bureau for Forensic Medical Expert Evaluations of the Leningrad region, are described. Unlike other methods used in Russia, this method detects the semen in stains in the absence of spermatozoa and in stains with very low amount of the semen. Our modification allows objective recording of the results with computer processing. The method is cheaper than its foreign analogs and its sensitivity is similar to them.     

Detection of human proteins in buried blood using ELISA and monoclonal antibodies: towards the reliable species indentification of blood stains on buried material.

The survival of human proteins in blood stains on fragments of cloth buried in exposed soil was examined in a 15-month investigation carried out from September 1990 to December 1991. During this period there was a wide variety of weather conditions. Samples were exhumed at 4-weekly intervals for 16 weeks and finally at 65 weeks; extracts of the stains were tested for albumin and IgG using a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) performed with monoclonal antibodies. Human albumin survived well throughout the 15 months of study, but IgG could be detected only in the 4- and 8-week samples. The reactions for IgG were weaker than those for albumin, although the method's sensitivity (10 ng) was the same for each protein. Appropriate buried and non-buried control experiments were carried out using cloth, either unstained or stained with human blood or animal sera; there was no cross-reactivity between human and the other species investigated and soil did not affect the assay; under laboratory conditions, IgG and albumin survived equally well. The system's versatility was illustrated by using monoclonal anti-bovine-albumin to detect specific albumin in the extracts of buried cloth which has been stained with bovine serum. It was concluded that ELISA performed with monoclonal antibodies could be of great value in identifying blood stains for forensic purposes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.     

An extremely sensitive species-specific ARMs PCR test for the presence of tiger bone DNA

The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for Traditional Chinese Medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed, if legislation prohibiting the trade in endangered species is to be enforced. A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other CITES protected species providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.     

Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy

Non-destructive identification and subsequent age estimation of blood stains are significant steps in forensic casework. The latter can provide important information on the temporal aspects of a crime. As previously shown, visible spectroscopy of blood stains on white backgrounds can successfully be used for their identification and age estimation. The use of this technique however, is hampered by dark backgrounds. In the present study the feasibility to use near infrared (NIR) spectroscopy was evaluated for blood stain identification and age estimation on dark backgrounds. Using NIR reflectance spectroscopy, blood stains were distinguished from other substances with 100% sensitivity and 100% specificity. In addition, Partial Least Squares Regression analysis was applied to estimate the age of blood stains on colored backgrounds. The age of blood stains up to 1 month old was estimated successfully with a root mean squared error of prediction of 8.9%. These findings are an important step toward the practical implementation of blood stain identification and age estimation in forensic casework, where a large variety of backgrounds can be encountered.     

An extremely sensitive species-specific ARMS PCR test for the presence of tiger bone DNA

The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for traditional Chinese medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed if legislation prohibiting the trade in endangered species is to be enforced.A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) protected species, providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.     

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.     

DNATyper^TM15试剂盒的法医学应用研究

目的研究DNATyper^TM15试剂盒在法医学中的应用。方法共收集60种8137份检材,在34个实验室,用5种不同方法提取DNA,使用DNATyperTM15试剂盒与2种同类进口试剂扩增并对比检测。结果DNATyperTM15试剂盒对不同提取方法、检材种类、扩增与检测仪器、操作环境、操作人员等均具有适应性。结论DNATyperTM15试剂盒能够用于法医学检验鉴定。     

Forensic scatology: preliminary experimental study of the preparation and potential for identification of captive carnivore scat

Carnivore scats recovered from animal attack and/or scavenging contexts frequently contain forensic evidence such as human bone fragments. Forensic cases with carnivore involvement are increasingly prevalent, necessitating a methodology for the recovery and analysis of scat evidence. This study proposes a method for the safe preparation of carnivore scat, recovery of bone inclusions, and quantification and comparison of scat variables. Fourteen scats (lion, jaguar, lynx, wolf, and coyote) were prepared with sodium-acetate-formalin fixative; analytical variables included carnivore individual, species, body size, and taxonomic family. Scat variables, particularly bone fragment inclusions, were found to vary among carnivore individuals, families, species, and sizes. The methods in this study facilitate safe scat processing, the complete recovery of digested evidence, and the preliminary identification of involved animals. This research demonstrates that scat collected from forensic contexts can yield valuable information concerning both the victim and the carnivore involved.     

Interpretation of ante-mortem stature estimates in South Africans

We developed a simple method for animal species identification of humans, dogs and cats, using a multiplex single-base primer extension reaction in the cytochrome b gene. Using this method, three points of a single nucleotide in the cytochrome b gene were examined in these species using primers of different lengths. Our method was found to be able to successfully identify humans (26 samples), dogs (21 samples) and cats (9 samples), and no differences were found among the samples from each animal species in this study. The amount of template DNA required was over 0.01 ng for humans and dogs, and over 0.1 ng for cats. The present method was able to identify animal species from hair shaft (2 cm) and forensic casework samples (blood stains and hair shafts), and is thus a useful tool for animal species (human, dog and cat) identification in forensic science.     

Profiler Plus试剂盒扩增Amelogenin基因座变异1例

目的探讨日常检案及DNA数据库建设中常用的ProfilerPlus系统扩增法医生物检材可能出现的变异情况。方法分别采用ProfilerPlus试剂盒扩增、毛细管电泳分析及Amelogenin单基因座扩增、银染技术分型两种方法分别检测两份血痕的基因型。结果应用ProfilerPlus试剂盒扩增、毛细管电泳分析发现X-Y同源Amelogenin基因座的X片段丢失,而运用Amelogenin单基因座扩增、银染技术分型则X片段正常出现。结论应用ProfilerPlus系统分析法医生物检材,有可能出现等位基因丢失现象,此现象需要引起我们在日常检案及DNA数据库建设中的足够重视。     

用引物Y_3,Y_4和DNA扩增技术作血液(痕)和毛根性别鉴定的研究

作者用引物Y_3、Y_4和DNA聚合酶链式反应(PCR)作微量人类血液(痕)和毛根的性别鉴定。扩增的靶序列位于Y染色体DNA特异3.4kb重复序列中,扩增产物为460bp。检材用量为:新鲜血液0.5μl、血痕纱纤维1mm、毛根单个。20例保存4个月的血痕与2例保存6年半的血痕性别判定结果均正确,无性别记载的保存9～11年的3例血痕显现了清晰的460bpY特异DNA扩增带。15例保存20天的自然脱落毛根性别判定结果均正确。本法省略了检材处理中的酚-氯仿抽提DNA等纯化步骤,既简化了实验操作,又减少了检验过程中外源DNA的污染机会和样品DNA的损耗,使这一性别鉴定方法更符合法医学实践的需要。     

An evaluation of gamma-glutamyl transpeptidase (GGT) and p30 determinations for the identification of semen on postcoital vaginal swabs

The following study entails the investigation of gamma-glutamyl transpeptidase (GGT) and p30 for the identification of seminal stains in sexual assault cases. A commercial kit was used to test for GGT activity, while p30 was demonstrated with a crossed electrophoresis technique. Specificity, sensitivity, and stability of both markers were studied. Postcoital swabs from lab staff were tested for GGT and p30. In addition, 144 postcoital swabs from case material were tested for p30, spermatozoa, and acid phosphatase. Results show p30 to be a useful semen marker particularly in cases of azoospermia. However, GGT was found to be unsuitable for forensic science casework.     

Development of a nanoplate-based digital PCR assay for species identification with mixture deconvolution

In crime scenes, not all biological stains are human in origin. Some exhibits can be from pets living on the premises or from animal products used in food consumption. In addition, it could be necessary to test animal carcasses for other forensic purposes. Often such stains can include mixtures involving humans or other species. Thus, identifying and deconvoluting mixtures of species commonly found in and around a household can be crucial in forensic casework. Different molecular techniques have been employed for species identification such as immunoprecipitation, qPCR, and DNA sequencing.In this project, a nanoplate-based digital PCR assay for species identification was developed, targeting Homo sapiens, canine, feline, bovine swine, pisces, and gallus in two multiplexes. An internal positive control was included in the design. The assay is simple, rapid, and can determine a wide variety of different vertebrates from biological exhibits, as well as in mixtures. Because the assay utilizes digital PCR, the procedure shows sensitivity down to a few copies, even in the presence of larger amounts of a major contributor, making the assay particularly useful in mixture deconvolution. Overall, this assay presents the forensic community with a novel application in which digital PCR can provide a sensitive and specific determination of species.     

Status and prospects of developing the Forensic Medical Service in the Russian Federation

Forensic medical service is constantly updated, which is confirmed by introduction of new modern methods, such as detection of blood or semen with test strips, new modifications of methods for handling bone material, differentiation of blood of the same group within ABO system, identification of blood groups in mixed stains of blood of humans and some animals, use of gel plates, etc. Methodological and information letters have been issued and field seminars are regularly carried for upgrading expert biologists. The overwhelming majority of expert biologists have received specialized education and topical upgrading.     

法医骨组织学研究

在实际检案中 ,当现场发现的骨骼残片体积较小时 ,用解剖学观察无法进行骨骼残片的法医鉴定 ,需使用骨组织学的方法进行骨骼残片的个体识别。目前 ,这是法医人类学中一门较活跃的领域 ,即法医骨组织学。法医骨组织学的内容主要包括两个方面 :(1)骨骼残片是否属于人类骨骼 ,或属于何种动物骨骼。这方面的研究包括人类骨骼的组织学特征研究及不同动物的组织学特征研究。 (2 )人类骨骼个体识别的组织学研究。这方面的研究主要包括 ,人类骨骼的组织学特征的年龄判断 ,例如股骨、胫骨、肱骨、锁骨等 ,以及使用骨组织学方法 ,进行人类骨骼的年龄评价的准确性研究。本文对上述内容进行了综述。     

Validation studies of an immunochromatographic 1-step test for the forensic identification of human blood.

An immunochromatographic 1-step test for the detection of fecal occult blood was evaluated for applicability for the forensic identification of human blood in stained material. The following experiments were conducted: 1) determination of the sensitivity and specificity of the assay; 2) evaluation of different extraction media for bloodstains (sterile water, Tris buffer pH 7.5 provided in the test kit, 5% ammonia); 3) analysis of biological samples subjected to a variety of environmental insults; and 4) evaluation of casework samples. This immunochromatographic 1-step occult blood test is specific for human (primate) hemoglobin and is at least an order of magnitude more sensitive than previous methods for detecting human hemoglobin in bloodstains. The antigen is insensitive to a variety of environmental insults, except for exposure to certain detergents and household bleaches and prolonged exposure to certain preparations of luminol. The entire assay can be conducted in field testing conditions within minutes. When in the laboratory the supernatant from a DNA extraction is used for the assay, there is essentially no consumption of DNA for determining the presence of human hemoglobin in a forensic sample. The data demonstrate that this test is robust and suitable for forensic analyses.     

ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.     

Identification of fetal blood stains by radioimmunoassay of alpha 1-fetoprotein.

Radioimmunoassay of alpha 1-fetoprotein(AFP) for medico-legal identification of fetal blood stains using a commercial kit is described. The AFP content in fetal blood stains on filter paper ranged from 21--320 ng/9 mm2. The protein was detected in stains of adult blood and retroplacental blood in only negligible amounts. Aging of the blood stains did not influence the values up to 1 month. The method is simple and sensitive enough for application to medico-legal-practice.