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.     

The ultrastructure of tissue attached to telogen hair roots

Most tissues encountered in forensic biology laboratories have been previously characterized with electron microscopy due to their medical importance. Anagen hair root cells, epithelial cells, erythrocytes, neutrophils, osteocytes, and spermatozoa have received considerable research attention at the ultrastructural level. There is no literature indicating that cells attached to removed telogen hair roots have been characterized with transmission electron microscopy. Nonetheless, telogen hairs are a frequent submission item to forensic laboratories for DNA typing. The amount of tissue attached to a telogen hair root usually determines whether that hair is suitable for nuclear DNA typing methods or mitochondrial DNA typing methods. This study used transmission and scanning electron microscopy to characterize the tissues found in three commonly occurring telogen hair root forms. The tissues were found to consist of keratinized remnant follicle, nonnucleated epithelial cells, nucleated epithelial cells, and trichilemmal keratin. These findings were consistent with the known principles of hair follicle regression. The recognition of the root structures that contain these specific tissue types may assist in the DNA typing of telogen hairs inasmuch as the quality of tissue present may be more important than the amounts of tissue present.     

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 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 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.     

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.     

The visualization and quantification of cell nuclei in telogen hair roots by fluorescence microscopy, as a pre-DNA analysis assessment

Although nuclear DNA-profiling of human hairs is a well-known technique in forensic investigations, its success rate is quite low. Because the extracted nuclear DNA (nuDNA) is scarce and often degraded, a simple and effective method was developed to estimate the number of cell nuclei in telogen roots. DAPI, a fluorescent, non-destructive DNA-stain, allows visualizing nuclear DNA and does not interfere with subsequent PCR analyses. After staining 3242 roots from 27 volunteers and subsequent STR-profiling of a selection of roots, we show that the amount of analysable nuDNA can be predicted. This screening method allows the genetic laboratory to analyse only the most promising hair roots.     

Preliminary study of hair form of Japanese head hairs using image analysis

The use of average curvature measurements for the forensic comparison of curly hairs has been reported, but a method, in which various types of hair form are quantitatively examined and objectively interpreted for hair comparison, has not been reported to date. In the present study, numerical data on hair form from Japanese subjects were obtained by image analysis and a morphological comparison of these head hairs was investigated. Head hairs obtained from eight Japanese males were measured for length (L), distance (D) and area (A) using a Kontron Imaging System KS400. From the three measurements mentioned above, three indexes, L/D, A/D and 2(A/L), were examined. The inter-individual variations for each value were investigated by a t-test and the availability of six values for the forensic comparison of hair form was evaluated by a stepwise linear discrimination analysis. Six values obtained from hair form by an image analysis showed large intra-individual variations. However, these six values were found to be useful for discriminating between two individuals, since the six values showed larger inter-individual variations than intra-individual variations. Discrimination on each comparison using a stepwise linear discrimination analysis was performed for some of the values and the results indicated conspicuous inter-individual variations between the two individuals. On 11 of 28 comparisons, 30 hairs from one individual could be completely distinguished from hairs of another individual, when a two-way comparison was employed. These results confirm that hair form could be quite useful in the forensic comparison of hair morphology, and suggest that numerical data obtained from hair form by image analysis are very important values for constructing a screening procedure for evidential hairs. The use of an objective measure of hair form will be especially useful for Japanese head hairs since they are generally thought to show very limited variation in morphological features.     

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.     

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.     

Individual identification of cats and dogs using mitochondrial DNA tandem repeats?

Cats and dogs are very common animals in the human environment. In Switzerland, one in five households owns a cat or a dog. Their hairs are very easily transferred and could be used as a frequent trace evidence. Using DNA analysis, identification of these animals should be possible as it is in human identification. However, most of the time, no nuclear DNA can be recovered from the hair. It is therefore necessary to rely on mtDNA. Cats and dogs have tandemly repeated sequences in their mtDNA control region. In this study, the authors show that these tandem repeats are very polymorphic but are also the source of a very high level of heteroplasmy. The authors, therefore, examined if this might prevent their use in forensic identification.     

The persistence of human scalp hair on clothing fabrics

This study reports the persistence behaviour of human scalp hairs under a number of different circumstances. The effects of artificial dyeing of hairs, the presence or absence of roots and different types of fabrics on the persistence of hair on a variety of garments were investigated. The garments were made from cotton, polycotton, cotton/acrylic, polyester and wool. The results indicated that neither artificial dyes nor the presence or absence of roots had statistically significant effects on the persistence of hair. In contrast, the type of fabric had a major impact and it was found that, generally, hairs persist longer on rougher fabrics. The rate of loss of hairs from non-woollen fabrics during normal wear was found to follow an exponential decay curve. In contrast, the rate of loss from the woollen garments was quite linear, indicating a constant, even loss, and thus suggests that a different process is involved in the persistence of hairs on woollen garments from that on non-woollen garments. The speed at which hair was lost from fabrics decreased in the order polyester, cotton/acrylic, polycotton, cotton, smooth wool, rough wool, so that wool gives the best chance of recovering samples of hair. Due to the uniqueness of each case, it is advised that caution be used when making any interpretations and before drawing any conclusions.     

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.     

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.     

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。     

Morphology of hairs on the head and other parts of the body in the residents of Africa

The morphology of hairs from the head, beard, chest, armpits, and pubis was studied in indigenous population of Sudan, Sierra-Leone, Cote d'Ivoir, Ruanda, Nigeria, Gambia, Kenya, Uganda, Burundi, Zaire, Southern African Republic, Angola, Benin, and Guinea Bissau. A total of 327 hair specimens were examined. A number of new parameters of hairs were determined: thickness, number of cuticle pattern lines per mm, optic edge pattern, cuticle patterns, methods for detecting the cortical and medullar layers, width of hair layers and their transverse sections. The resultant morphological signs are new specific signs for African residents (Negroid) which can be used in forensic medical studies for identifying the race appurtenance of hairs.     

烫发、梳理和拉伸损伤对头发角蛋白的影响

目的为法医物证学中的头发个人识别提供依据。方法采用烫发、梳理、拉伸等方式对正常人头发进行损伤处理,用十二烷基磺酸钠-聚丙烯酰胺凝胶梯度电泳(SDS-PAGGE)和激光光密度扫描仪对损伤头发角蛋白进行分析。结果三种损伤条件均可导致头发损伤,造成分子量(MW)67000～43000区域头发角蛋白的丢失。结论头发角蛋白的丢失程度随着损伤程度增加而增加。     

The use of microscopic postmortem changes in anagen hair roots to associate questioned hairs with known hairs and reconstruct events in two murder cases

In two cases investigated by the New Orleans Police Department Crime Lab, hairs recovered from crime scenes were found to exhibit microscopic postmortem changes in anagen hair roots. These microscopic characteristics were used to associate these hairs with various victims in the cases. In addition to associating questioned hairs with known hairs, the fact that the victims were dead when the hairs were pulled helped investigators reconstruct events in both crimes and corroborate statements made by the arrested subjects in each case.