Nuclear and mitochondrial DNA quantification of various forensic materials

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.     

Human hair histogenesis for the mitochondrial DNA forensic scientist.

Analysis of mitochondrial DNA (mtDNA) sequence from human hairs has proven to be a valuable complement to traditional hair comparison microscopy in forensic cases when nuclear DNA typing is not possible. However, while much is known about the specialties of hair biology and mtDNA sequence analysis, there has been little correlation of individual information. Hair microscopy and hair embryogenesis are subjects that are sometimes unfamiliar to the forensic DNA scientist. The continual growth and replacement of human hairs involves complex cellular transformation and regeneration events. In turn, the analysis of mtDNA sequence data can involve complex questions of interpretation (e.g., heteroplasmy and the sequence variation it may cause within an individual, or between related individuals. In this paper we review the details of hair developmental histology, including the migration of mitochondria in the growing hair, and the related interpretation issues regarding the analysis of mtDNA data in hair. Macroscopic and microscopic hair specimen classifications are provided as a possible guide to help forensic scientists better associate mtDNA sequence heteroplasmy data with the physical characteristics of a hair. These same hair specimen classifications may also be useful when evaluating the relative success in sequencing different types and/or forms of human hairs. The ultimate goal of this review is to bring the hair microscopist and forensic DNA scientist closer together, as the use of mtDNA sequence analysis continues to expand.     

Microscopical discrimination of human head hairs sharing a mitochondrial haplogroup

In forensic analyses, determining the level of consensus among examiners for hair comparison conclusions and ancestry identifications is important for assessing the scientific validity of microscopical hair examinations. Here, we present data from an interlaboratory study on the accuracy of microscopical hair comparisons among a subset of experienced hair examiners currently analyzing hair in forensic laboratories across the United States. We examined how well microscopical analysis of hair can reliably be used to differentiate hair samples, many of which were macroscopically similar. Using cut hair samples, many sharing similar macroscopic and microscopic features, collected from individuals who share the same mitochondrial haplogroup as an indication of genetic relatedness, we tested multiple aspects that could impact hair comparisons. This research tested the extent to which morphological features related to ancestry and hair length influence conclusions. Microscopical hair examinations yielded accurate assessments of inclusion/exclusion relative to the reference samples among 85% of the pairwise comparisons. We found shorter hairs had reduced levels of accuracy and hairs from populations examiners were not familiar with may have impacted their ability to resolve features. The reliability of ancestry determinations is not yet clear, but we found indications that the existing categories are only somewhat related to current ethnic and genetic variation. Our results provide support for the continued utility of microscopical comparison of hairs within forensic laboratories and to advocate for a combined analytical approach using both microscopical analysis and mtDNA data on all forensic analyses of hair.     

Microscopical discrimination of twins' head hair

Twin populations are ideal for studying human variation; a study of twin's hair, therefore, provided a better understanding of the value of hair comparisons. Duplicate head hair samples from 17 pairs of twins and one set of identical triplets were compared in a verified blind study. In addition to the direct comparison of all twins, random samples of two or three hairs were compared with randomly selected groups of known samples in a second blind study, to better simulate an ordinary forensic science case. Features commonly used by forensic hair examiners were adequate to distinguish hair samples from each twin from all other samples, illustrating the power of microscopical comparison when numerous questioned hairs are available in evidence. When two or three hairs were compared with randomly selected known samples, several were indistinguishable from hair samples other than the true source, proving once again that a human hair can never be associated with one person to the exclusion of all others.     

毛发中毒品分析

毛发分析在法庭毒品分析领域有其独特的优势,很多国家的法化学实验室,毛发分析已成为毒品检测的常规操作,并已得到了法庭的承认、采纳。本文对毒品进入毛发的机制、毛发的现场勘查、毛发中毒品分析程序、毛发中毒品分析结果的评判进行了综述。简单地介绍了毛发在现场勘查中采取、包装、送检的基本方法和技术人员在操作过程中的注意事项,以及针对毛发检验中的特殊技术处理;另外,介绍了毛发中毒品分析的特点,通过分析毛发毒品的药理机制,总结出了一些高效、便利、快速的毒品分析方法,并对各种方法进行了介绍,得出了毛发中毒品分析结果的一些特点。     

A comparison study of hair examination methodologies

A study was conducted to investigate the accuracy between two methods of hair analysis: PCR-STR DNA analysis and microscopic comparison analysis. Standard sets of pubic hairs were collected from volunteers, and unknown sets were generated from these samples. Three out of five (60%) of the hairs analyzed produced full DNA profiles that were correctly matched to the standard sets. DNA analysis was inconclusive (partial or no DNA profile) for two out of five (40%) of the samples. In contrast, the microscopic comparison analysis correctly matched four out of five (80%) of the samples to the standard sets but mis-identified one out of five (20%) of the samples. These results reinforce the practice of preliminary microscopic hair examination in narrowing down a set of hairs for DNA analysis. Microscopic comparison analysis is sufficiently reliable to remain a rapid and inexpensive method for forensic hair analysis.     

Mitochondrial DNA sequencing of human hair shafts stored for long time

Mitochondrial DNA (mtDNA) sequencing is commonly used for forensic genetic identification of relation and personality identification based on analysis of tooth and skeletal rudiments. We demonstrated the possibility of DNA extraction and subsequent enzymatic amplification of fragments of a hypervariable segment I of mtDNA control region from hair shafts after long storage (up to 75 years). Shed hairs are the most common biological material evidence in forensic investigations. Low content of DNA and its possible degradation in hair shafts without bulbs may cause artifacts in polymerase chain reaction. However comparative analysis of amplified nucleotide sequences of amplified fragments from hair stored for 75 years was identical to the sequence from hairs cut immediately before experiment. This indicates high quality of the resultant matrices, stability of results, and hence, the possibility of using DNA extracted from hair shafts without bulbs stored for a long time for expert genetic analysis. Theoretical and methodological prospects of using mtDNA polymorphism analysis for forensic expert evaluations are discussed.     

A Novel Application of a Cryosectioning Technique to Aid Scat Hair Microanalysis

Scat hair presents a diverse profile of hairs for morphological assessment that may find versatile applications in wildlife forensic investigations. Successful morphological assessment of scat hair microstructure, however, depends on a robust sectioning methodology. We assessed the feasibility and efficacy of a cryosectioning technique compared to that of a gold standard hand‐sectioning technique. Scat hairs were embedded in paraffin wax and hand‐sectioned, while cryopreserved scat hairs were sectioned with a cryostat. The results showed that cryosectioning preserved the pristine morphology of the scat hair and provided cross sections more amenable to high‐resolution imaging of hair internal microstructure than hand‐sectioning. The cryosectioning technique may find novel applications as a more reliable and robust technique to aid (i) scat hair internal microstructure analysis for cross‐referencing with species identification keys in wildlife forensic studies and (ii) downstream toxicological analysis in wildlife forensic studies as hair biochemistry is not altered during cryopreservation.     

Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification

The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification.     

脱落毛发线粒体DNA HV1区序列测定的研究

目的　对脱落毛发线粒体DNAHV1区序列测定方法进行研究。方法　嵌合扩增结合末端荧光标记DNA测序。结果　对 2 0例脱落毛发进行分析获得了明确的测序结果 ,与来自同一个体的血液所测得的DNA序列进行比较 ,完全相同。结论　嵌合扩增在对脱落毛发进行线粒体DNA多变区序列分析中是一种有效的方法 ,在法医DNA检验中具有实用价值。     

STR typing of ancient DNA extracted from hair shafts of Siberian mummies

The aim of this study was to determine if ancient hair shafts could be suitable for nuclear DNA analysis and to develop an efficient and straightforward protocol for DNA extraction and STR typing of ancient specimens. The developed method was validated on modern and forensic samples and then successfully applied on ancient hairs collected from Siberian mummies dating from the 16th to the early 19th centuries. In parallel extractions including or excluding a washing step were performed at least two times for each sample in order to evaluate the influence on the quantity of nuclear DNA yielded and on the typing efficiency. Twelve ancient individuals were analyzed through our approach and full and reliable profiles were obtained for four of them. These profiles were validated by comparison with those obtained from bone and teeth DNA extracted from the same ancient specimens. The present study demonstrates that the washing step cannot be considered as deleterious for DNA retrieval since the same results were obtained by the two approaches. This finding challenges the hypothesis that recoverable nuclear DNA is only found on the outer surface of hair shafts and provides evidence that nuclear DNA can be successfully extracted from ancient hair shafts. The method described here constitutes a promising way for non-invasive investigations in ancient DNA analysis for precious or historical samples as well as forensic casework analyses.     

Investigating the morphology and genetics of scalp and facial hair characteristics for phenotype prediction

Microscopic traits and ultrastructure of hair such as cross-sectional shape, pigmentation, curvature, and internal structure help determine the level of variations between and across human populations. Apart from cosmetics and anthropological applications, such as determining species, somatic origin (body area), and biogeographic ancestry, the evidential value of hair has increased with rapid progression in the area of forensic DNA phenotyping (FDP). Individuals differ in the features of their scalp hair (greying, shape, colour, balding, thickness, and density) and facial hair (eyebrow thickness, monobrow, and beard thickness) features. Scalp and facial hair characteristics are genetically controlled and lead to visible inter-individual variations within and among populations of various ethnic origins. Hence, these characteristics can be exploited and made more inclusive in FDP, thereby leading to more comprehensive, accurate, and robust prediction models for forensic purposes. The present article focuses on understanding the genetics of scalp and facial hair characteristics with the goal to develop a more inclusive approach to better understand hair biology by integrating hair microscopy with genetics for genotype-phenotype correlation research.     

Correlation of microscopic and mitochondrial DNA hair comparisons

Expert opinions regarding the microscopic comparison of human hairs have been accepted routinely in courts for decades. However, with the advent of mitochondrial DNA (mtDNA) sequencing, an assessment can be made of the association by microscopic hair comparisons in casework between a questioned hair and reference hairs from an individual. While each method can be used separately, the two analytical methods can be complementary and together can provide additional information regarding source association. Human hairs submitted to the FBI Laboratory for analysis between 1996 and 2000 were reviewed. Of 170 hair examinations, there were 80 microscopic associations; of these, only nine were excluded by mtDNA. Importantly, 66 hairs that were considered either unsuitable for microscopic examinations or yielded inconclusive microscopic associations provided mtDNA results. Only six hairs did not provide sufficient mtDNA, and only three yielded inconclusive results. Consistency was observed in exculpatory results with the two procedures. This study demonstrates the utility of microscopic hair examinations and the strength of combining microscopic analysis with mtDNA sequencing.     

Hair-MAP: a prototype automated system for forensic hair comparison and analysis

This paper demonstrates the feasibility of the automation of forensic hair analysis and comparison task using neural network explanation systems (NNESs). Our system takes as input microscopic images of two hairs and produces a classification decision as to whether or not the hairs came from the same person. Hair images were captured using a NEXTDimension video board in a NEXTDimension color turbo computer, connected to a video camera. Image processing was done on an SGI indigo workstation. Each image is segmented into a number of pieces appropriate for classification of different features. A variety of image processing techniques are used to enhance this information. Use of wavelet analysis and the Haralick texture algorithm to pre-process data has allowed us to compress large amounts of data into smaller, yet representative data. Neural networks are then used for feature classification. Finally, statistical tests determine the degree of match between the resulting collection of hair feature vectors. An important issue in automation of any task in criminal investigations is the reliability and understandability of the resulting system. To address this concern, we have developed methods to facilitate explanation of neural network's behavior using a decision tree. The system was able to achieve a performance of 83% hair match accuracy, using 5 of the 21 morphological characteristics used by experts. This shows promise for the usefulness of a fuller scale system. While an automated system would not replace the expert, it would make the task easier by providing a means for pre-processing the large amount of data with which the expert must contend.     

Development of a Protein‐based Human Identification Capability from a Single Hair

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high‐resolution nanoflow liquid chromatography‐based mass spectrometry and a novel exome‐driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10^?2 and 6.0 × 10^?9. The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.     

A review of major factors contributing to errors in human hair association by microscopy.

Forensic hair examiners using traditional microscopic comparison techniques cannot state with certainty, except in extremely rare cases, that a found hair originated from a particular individual. They also cannot provide a statistical likelihood that a hair came from a certain individual and not another. There is no data available regarding the frequency of a specific microscopic hair characteristic (i.e., microtype) or trait in a particular population. Microtype is a term we use to describe certain internal characteristics and features expressed when observing hairs with unpolarized transmitted light. Courts seem to be sympathetic to lawyer's concerns that there are no accepted probability standards for human hair identification. Under Daubert, microscopic hair analysis testimony (or other scientific testimony) is allowed if the technique can be shown to have testability, peer review, general acceptance, and a known error rate. As with other forensic disciplines, laboratory error rate determination for a specific hair comparison case is not possible. Polymerase chain reaction (PCR)-based typing of hair roots offer hair examiners an opportunity to begin cataloging data with regard to microscopic hair association error rates. This is certainly a realistic manner in which to ascertain which hair microtypes and case circumstances repeatedly cause difficulty in association. Two cases are presented in which PCR typing revealed an incorrect inclusion in one and an incorrect exclusion in another. This paper does not suggest that such limited observations define a rate of occurrence. These cases illustrate evidentiary conditions or case circumstances which may potentially contribute to microscopic hair association errors. Issues discussed in this review paper address the potential questions an expert witness may expect in a Daubert hair analysis admissibility hearing.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

Forensic hair morphology comparison--a dying art or junk science?

There has been debate in both the judicial and forensic fields concerning the admissibility and reliability of the so-called forensic comparison sciences such as handwriting, tool mark analyses, and hair analysis. In particular, there has been increasing controversy over the use and interpretation of hair comparison evidence and it has been held partly responsible for miscarriages of justice. There has also been a perceived devaluation of the worth of microscopic human hair analysis particularly since the advent of DNA profiling. This article will attempt to initiate discussion on the past, current and future role of forensic human hair analysis and comparison.     

Statistical comparison of dog and cat guard hairs using numerical morphology

Numerical features obtained from the guard hairs of dogs and cats (total 300 hairs per dog or cat) were statistically compared, in an attempt to discriminate between them. Using hairs from each of five mongrel dogs and cats, eight measurements (length (Len), maximum width (MaxWid), cross sectional maximum diameter, cross sectional minimum diameter, cuticular thickness of the cross section and three scale counts per 100 microm length (observed at three positions: distal third (disSC), middle (midSC) and the proximal third (proSC) portions) and five indexes (hair width index (HWI), medulla index (MI), hair index, cuticle index and the difference in scale counts between the distal and proximal parts (defSC)) were examined. The range for each numerical feature overlapped each other extensively, and none of the features permitted a discrimination between dog and cat hairs, based on the values obtained. However, 12 numerical features, except for the midSC, showed a statistically significant difference between dog and cat hairs, as evidenced by a t-test. For the purpose of comprehensively comparing numerical features and statistically discriminating between dog and cat hairs, a discriminant analysis between the two were carried out using a multiple regression analysis. Four types of discriminant functions produced by combining over five numerical features were examined. Dog and cat hairs could clearly be discriminated using any of the discriminant functions. Species discrimination using the discriminant function permitted the species of a dog or cat to be determined, based on the overall morphologies of various numerical features. When experimentally collected test samples were investigated using the discriminant function using Combination-2, consisting of eight numerical features (Len, MaxWid, MI, HWI, disSC, midSC, proSC and defSC), all 10 cat hairs were correctly determined to be cat hair and 22 of 23 dog hairs were correctly identified. This discriminant function produced good results for species discrimination between dog and cat hairs.