A comparison of the sensitivity of typing group-specific component (Gc) and the phosphoglucomutase (PGM1) locus in control and casework bloodstains by three isoelectric focusing methods

The performance of typing group-specific component (Gc) in bloodstains by two isoelectric focusing methods followed by its detection with silver staining has been compared with an established forensic system of typing phosphoglucomutase (PGM1) locus phenotypes by isoelectric focusing (IEF) in 1 mm gels. For Gc typing ultra-thin isoelectric focusing (UTIEF) gels and immobilized pH gradient (IPG) gels were used. Both laboratory prepared stains and casework stains were examined. The Gc UTIEF method is approximately eight times more sensitive than the existing PGM1 1 mm IEF method for control and casework stains. However, on average, a larger amount of stain was taken from casework stains than control stains for each typing system. A total of 53 casework stains were examined. Comparable success rates of 62% and 64% were obtained for typing Gc on UTIEF gels and PGM1 by 1 mm IEF, respectively. A success rate of 55% was obtained for typing Gc on IPGs. Bloodstains that were over 200 days old were successfully grouped by all three methods.     

Identification of fetal hemoglobin in blood stains by high performance liquid chromatography

A new method for the identification of fetal hemoglobin (Hb F) in blood stains by reverse-phase high performance liquid chromatography is described. Differentiation between fetal and adult blood stains is based on the existence of gamma-chain peaks which are characteristics of Hb F. Very few gamma chains appeared on chromatograms of all the adult blood stains examined. The level of Hb F could be determined by measuring the total of chromatogram gamma-globin chain areas, and expressing it as a percentage of total Hb. Levels in six cord blood stains on filter paper ranged from 81.1% to 91.3% and remained constant for at least 12 weeks. This method is of great value for its simplicity, sensitivity and speed, and most importantly for its reliability in the field of forensic medicine.     

Identification of semen in stain by determination of the specific activity of L-tartrate-inhibitable acid phosphatase

For identification of semen in stain the specific activity of L-tartrate-inhibitable acid phosphatase (ACP) was determined. With each stain extract, both enzyme activity and protein concentration were determined, and the specific activity (enzyme activity/protein concentration) was calculated. Seminal stains showed a value of 23.8 +/- 15.2 (mean +/- SD) IU/mg protein, while vaginal fluid stains showed a value of 0.088 +/- 0.049 IU/mg protein. Stains of other body fluids did not show any L-tartrate-inhibitable ACP activity. Furthermore, only eight of 30 plant juice stains showed any levels of L-tartrate-inhibitable ACP, although all plants tested showed ACP activity. As the present method enables us to analyze forensic samples quantitatively, it seems to be useful for forensic practice.     

Possibility of the species identification using blood stains located on the material evidences and bone fragments with the method of solid phase enzyme immunoassay with "IgG general-EIA-BEST" kit and human immunoglobulin G

The authors give the comparative analysis of Russian and foreign forensic medical methods of species character identification of the blood from the stains on the material evidences and bone fragments. It is shown that for this purpose it is feasible to apply human immunoglobulin G (IgG) and solid phase enzyme immunoassay (EIA) with the kit "IgG general-EIA-BEST". In comparison with the methods used in Russia this method is more sensitive, convenient for objective registration and computer processing. The results of experiments shown that it is possible to use the kit "IgG general-EIA-BEST" in forensic medicine for the species character identification of the blood from the stains on the material evidences and bone fragments.     

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.     

Forensic biological examination--the reality and perspectives

The point of view of authors on the modern condition and priority directions of the development of examination of biological material evidence is set out. The main idea consists in the necessarily of reorganization and correction of organization methodical base of examination of biological material evidence. The role and place in examination of material evidence of traditional serological and new--molecule genetic components are determined in this direction. It is noted that forensic biology must not ever be associated with only one of its part--serological methods of analysis. Under growing influence of molecule genetic component the semantic, and methodical content of forensic examination of material evidence. This is not already auxiliary differentiation of examination objects but conclusive set of their equality or difference. Thus forensic biological department of forensic medical examination Office must become expert compartments of new type due for complex analysis of material evidence where molecule genetic technologies lie in the centre. All other methodical compartments of forensic biology also will be used but as auxiliary and additional methods. In perspective it is necessary to enlarge and centralize of high-tech forensic molecule genetic laboratories based on common science-methodical approach. Unfavorable outcome of medical aid: study of the problem in forensic medicine practice     

Successful DNA typing of urine stains using a DNA purification kit following dialfiltration

To evaluate the utility of DNA polymorphism typing of urine stains in forensic investigations, the amplifiable amount of DNA was estimated in 20 urine specimens obtained from 10 male and 10 female volunteers using a DNA purification kit following dialfiltration. DNA obtained from both urine and urine stains was amplified with the AmpflSTR Profiler PCR Amplification Kit, and was analyzed by capillary electrophoresis using the Genetic Analyzer. The amount of male and female urine necessary for obtaining a complete DNA profile was 0.2 mL and 0.08 mL, respectively. When 0.2 mL of male urine were used to create urine stains, complete DNA profiles could be obtained from just some of the stains. However, when only 0.1 mL of female urine was used, complete profiles could be successfully obtained from all of the stains. DNA on bleached cotton remained amplifiable for 3-6 weeks. This method using a DNA purification kit following dialfiltration can be recommended for the genotyping of urine stains.     

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.     

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.     

人像检验鉴定探讨

当前通过人像检验认定是否为同一人的应用需求日趋增加,但由于人像检验较为复杂、主观性较强、缺少普遍接受的方法等原因,一定程度上影响了人像检验鉴定的开展。为进一步明晰人像检验鉴定相关问题,本文就人像检验鉴定关键问题进行梳理讨论,重点对国内外人像检验标准、人像特征、检验步骤和方法、检验结论等进行探讨,以期为更好地开展此项工作提供参考。     

Performance Study of a Score‐based Likelihood Ratio System for Forensic Fingermark Comparison

In this article, the performance of a score‐based likelihood ratio (LR) system for comparisons of fingerprints with fingermarks is studied. The system is based on an automated fingerprint identification system (AFIS) comparison algorithm and focuses on fingerprint comparisons where the fingermarks contain 6–11 minutiae. The hypotheses under consideration are evaluated at the level of the person, not the finger. The LRs are presented with bootstrap intervals indicating the sampling uncertainty involved. Several aspects of the performance are measured: leave‐one‐out cross‐validation is applied, and rates of misleading evidence are studied in two ways. A simulation study is performed to study the coverage of the bootstrap intervals. The results indicate that the evidential strength for same source comparisons that do not meet the Dutch twelve‐point standard may be substantial. The methods used can be generalized to measure the performance of score‐based LR systems in other fields of forensic science.     

In depth investigation of the capabilities and limitations of combined proteomic-MALDI MS based approach for the forensic detection of blood

For the past 7 years, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) based methods have been developed and published for the forensic detection of blood in stains and fingermarks. However, in the view of adoption in an operational context, further investigation into the capabilities and limitations of this approach must be conducted. The refinement and testing of this approach must also be tailored to the requirements of the end users, enabling them to address the specific circumstances most encountered in a forensic scenario. The present study delves deeper into the assessment of the applicability of MALDI MS based strategy for the reliable and robust detection of human blood through: (i) a semi-qualitative assessment of the sensitivity of the method, (ii) a wider investigation of the compatibility of the method with the prior application of commonly used presumptive tests and (iii) assessment of the specificity of the method (when blood is present in mixture with other biofluids) and of its robustness, by assessing blood detection from a range of porous materials. The findings strengthen the evidence supporting the adoption of MALDI MS based approaches as a confirmatory test for the forensic detection of human blood in an operational context.     

Validation of PrepFiler™ forensic DNA extraction kit (Applied Biosystems)

The PrepFiler™ is a new kit recently introduced by Applied Biosystems for DNA extraction from a wide range of forensic samples. In the present study we tested the performance of PrepFiler™ kit against other commonly used commercially available kits on a variety of real forensic casework samples: bloodstains on different substrates, washed bloodstains, semen stains, saliva stains, hairs, bones, tissues, nails, prints after chemical treatments, skin swabs.     

3种DNA提取法在污染严重混合斑分型中的应用比较

目的比较Chelex-100法、酚/氯仿法和二氧化硅膜法3种DNA提取法在污染严重混合斑分型中的应用效果。方法从日常案例中收集污染严重的混合斑25份,差异消化法分离精子后同时用Chelex-100法、酚/氯仿法和二氧化硅膜技术3种方法提取DNA,采用PCR-STR技术对D19S253、FGA和CSF1PO 3个基因座进行分型,Gel-Pro软件处理电泳图谱,SPSS软件分析比较不同方法之间的差异。结果采用Chelex-100法提取DNA,25份检材分型结果均未成功;采用酚/氯仿法,25份检材中10份分型成功,3份检材FGA和CSF1PO基因座可分型,4份检材CSF1PO基因座可分型;采二氧化硅膜纯化法,25份检材均成功分型;酚/氯仿法和二氧化硅膜法两种方法比较,结果存在显著性差异(P<0.05)。结论二氧化硅膜纯化技术可以有效去除PCR抑制物,提取的DNA扩增效果明显优于Chelex-100法和酚/氯仿法,具有较高的应用价值。     

HLA—A基因座分步PCR—SSP分型研究

目的使HLA基因分型能应用于法医常见检材的个人识别。方法 建立检测HLA—A基因座的分步PCR—SSP方法。先用一对HLA—A基因座特异的引物作第一次扩增,以所得产物为模板,分别用对HLA—A30、A31、A33特异的3对引物作第二次扩增,二次扩增的产物经电泳判型。结果 1130例血清分型为HLA—A30、A31、A33的血痕,其PCR—SSP分型和血清分型的不符合率为29％;室温保存2年的精斑、唾液斑,保存18年的血痕第一次扩增均获得满意的结果。结论法医亲子鉴定和个人识别宜用基因分型替代血清分型。HLA—A基因座分步PCR—SSP基因分型适用于法医检材。     

Assessment of the methodology for estimating ridge density in fingerprints and its forensic application

In recent times, some studies have explored the forensic application of dermatoglyphic traits such as the epidermal ridge breadth or ridge density (RD) toward the inference of sex and population from fingerprints of unknown origin, as it has been demonstrated that there exist significant differences of fingerprints between sexes and between populations. Part of the population differences found between these studies could be of methodological nature, due both to the lack of standardisation in the position of the counting area, as well as to the differences in the method used for obtaining the fingerprint. Therefore, the aim of this study was to check whether there are differences between the RD of fingerprints depending on where the counting area is placed and how the fingerprints are obtained. Fingerprints of each finger were obtained from 102 adult Spanish subjects (50 females and 52 males), using two methods (plain and rolled). The ridge density of each fingerprint was assessed in five different areas of the dactylogram: two closer to the core area (one on the radial and the other on the ulnar side), two closer to the outermost area of each of the sides (radial and ulnar), and another one in the proximal region of the fingertip. Regardless of the method used and of the position of the counting area, thumbs and forefingers show a higher RD than middle, ring, and little fingers in both sexes, and females present a higher RD than males in all areas and fingers. In both males and females, RD values on the core region are higher than those on the outer region, irrespective of the technique of fingerprinting used (rolled or plain). Regardless of the sex and location of the count area (core or outer), the rolled fingerprints exhibit RD greater than that of the plain ones in both radial and proximal areas, whereas the trend is inverted in the ulnar area, where rolled fingerprints demonstrate RD lesser than that of the plain ones. Therefore, in order for the results of different studies to be comparable, it is necessary to standardise the position of the count area and to use the same method of obtaining the fingerprint, especially when involving a forensic application.     

Testing the reliability of frontal sinuses in positive identification

The use of frontal sinus radiographs in positive identification has become an increasingly applied and accepted technique among forensic anthropologists, radiologists, and pathologists. From an evidentiary standpoint, however, it is important to know whether frontal sinus radiographs are a reliable method for confirming or rejecting an identification, and standardized methods should be applied when making comparisons. The purpose of the following study is to develop an objective, standardized comparison method, and investigate the reliability of that method. Elliptic Fourier analysis (EFA) was used to assess the variation in 808 outlines of frontal sinuses by calculating likelihood ratios and posterior probabilities from EFA coefficients. Results show that using EFA coefficient comparison to estimate the probability of a correct identification is a reliable technique, and EFA comparison of frontal sinus outlines is recommended when it may be necessary to provide quantitative substantiation for a forensic identification based on these structures.     

Estimating Individual Contributions to Complex DNA SNP Mixtures.,

High‐throughput sequencing (HTS) of large panels of single nucleotide polymorphisms (SNPs) provides an alternative or complimentary approach to short tandem repeats (STRs) panels for the analysis of complex DNA mixture forensic samples. For STRs, methods to estimate individual contribution concentrations compare capillary electrophoresis peak heights, peak areas, or HTS allele read counts within a mixture. This article introduces three approaches (mean, median, and slope methods) for estimating individual DNA contributions to forensic mixtures for HTS/massively parallel sequencing (MPS) SNP panels. For SNPs, the major:minor allele ratios or counts, unique to each contributor, were compared to estimate contributor proportion within the mixture using the mean, median, and slope intercept for these alleles. The estimates for these three methods were typically within 5% of planned experimental contributions for defined mixtures.     

混合血迹中不同个体成份逐一分离识别的研究

目的研究法庭科学混合血迹物证中不同个体成份逐一分离、识别的问题,建立适合混合血迹个体识别的分析技术。方法采用PCR-SSCP及测序技术,选择m tDNA D-loop区的HVI 16030~16481区域452 bp片段作为分析目标,对中国汉族两无关个体、三无关个体混合血迹进行分析。结果100份两个体混合血迹样品m tDNA 452bp的PCR产物经SSCP电泳分离,结果有95份样品完全分离开,分离成功率达95%;30份三个体混合血迹样品452 bp片段经SSCP电泳分离,结果有26份样品有1~3个个体完全分离开,分离成功率达84%。对其中3份两个体混合血样、2份三个体混合血样SSCP电泳分离后的谱带进行回收、测序分析,两个体混合血样每一份均可准确获得其中单一个体序列及以另一个体主要成份(峰值比达4∶1以上)的序列结果;三个体混合血迹中不同个体成份可以达到初步分离,1份可准确确定单一个体序列。对两个体不同比例混合样品SSCP分析,结果可以检测到较少成份的最低比例为20∶80。结论本研究建立的PCR-SSCP及测序分析混合血迹综合技术,是对混合血迹中不同个体成份逐一分离、识别的一种有效技术手段。