Differentiation of Morphologically Similar Human Head Hairs from Two Demographically Similar Individuals Using Amino Acid Ratios,

Human hair is frequently encountered as forensic evidence and can contribute valuable information to investigators. Conventional forensic hair analyses include microscopic hair comparison (MHC) and DNA analysis. However, MHC is not supported by statistics and DNA analysis cannot always be performed. Recent studies have demonstrated that evaluation of differences in the hair proteins may offer an alternate method to these analyses. In this study, an evaluation of the amino acids present in hair was investigated as an approach to differentiate morphologically indistinguishable hair samples from two demographically similar individuals. Proteins in the hair were digested using hydrochloric acid, and the resulting amino acids were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) for analysis using gas chromatography-mass spectrometry (GC-MS). Eight derivatized amino acids were detected and quantified relative to an internal standard, L-norvaline, and used to construct twenty-eight amino acid ratios. Hair samples were collected from four areas of the head on various days over the course of one month, and no significant differences in amino acid ratios (p-value > 0.05) were observed among the areas of the head, and the ratios were consistent over the time period of this study. Additionally, fifteen of these amino acid ratios were found to be significantly different between the two individuals when compared using a two-sample t-test (p-value ≤ 0.05). These data indicate that amino acid analysis was able to differentiate two morphologically similar hair samples from different individuals and demonstrates the applicability of this method to distinguish similar hair samples when DNA analysis cannot be performed.     

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.     

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.     

Sensitive Method for the Confident Identification of Genetically Variant Peptides in Human Hair Keratin

Recent reports have demonstrated that genetically variant peptides derived from human hair shaft proteins can be used to differentiate individuals of different biogeographic origins. We report a method involving direct extraction of hair shaft proteins more sensitive than previously published methods regarding GVP detection. It involves one step for protein extraction and was found to provide reproducible results. A detailed proteomic analysis of this data is presented that led to the following four results: (i) A peptide spectral library was created and made available for download. It contains all identified peptides from this work, including GVPs that, when appropriately expanded with diverse hair-derived peptides, can provide a routine, reliable, and sensitive means of analyzing hair digests; (ii) an analysis of artifact peptides arising from side reactions is also made using a new method for finding unexpected modifications; (iii) detailed analysis of the gel-based method employed clearly shows the high degree of cross-linking or protein association involved in hair digestion, with major GVPs eluting over a wide range of high molecular weights while others apparently arise from distinct non-cross-linked proteins; and (v) finally, we show that some of the specific GVP identifications depend on the sample preparation method.     

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.     

毒品与毛发的结合及其影响因素

毛发毒品的分析在毒品检验中具有独特优势,而毛发与毒品的结合状况及毒品聚集于毛发的机制影响其检测的准确性和灵敏性。对毒品与毛发的结合位点、毒品与毛发结合的差异性因素以及外界因素对毒品与毛发结合的影响进行了综述。     

Timing and Appearance of Postmortem Root Banding in Nonhuman Mammals,

A study was undertaken using nonhuman mammal specimens to better understand environmental influences on postmortem hair root band (PMRB) formation and to see whether PMRBs would occur in nonhuman mammal hairs in a similar fashion to human hairs. Carcasses from surrounding roadways were the primary source of specimens for this study, augmented by donated deceased domestic pets. Sections of pelt from each specimen were placed in controlled environmental conditions while the remainder of the carcass was left in a secure outdoor setting. Hair samples were collected daily from outdoor and control specimens and examined for evidence of PMRBs. Several environmental factors were also recorded on a daily basis. Results demonstrate PMRBs can occur in nonhuman mammal hairs, and they have microscopic characteristics similar to human PMRBs. Factors found to correlate with PMRB formation include postmortem interval, temperature, pH, and the formation and subsequent volatilization of ammonia from the surrounding tissue.     

Simultaneous Quantitative Determination of Amphetamines,Opiates, Ketamine,Cocaine and Metabolites in Human Hair: Application to Forensic Cases of Drug Abuse

A method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to simultaneously quantify amphetamines, opiates, ketamine, cocaine, and metabolites in human hair is described. Hair samples (50 mg) were extracted with methanol utilizing cryogenic grinding. Calibration curves for all the analytes were established in the concentration range 0.05–10 ng/mg. The recoveries were above 72%, except for AMP at the limit of quantification (LOQ), which was 48%. The accuracies were within ±20% at the LOQ (0.05 ng/mg) and between −11% and 13.3% at 0.3 and 9.5 ng/mg, respectively. The intraday and interday precisions were within 19.6% and 19.8%, respectively. A proficiency test was applied to the validated method with z-scores within ±2, demonstrating the accuracy of the method for the determination of drugs of abuse in the hair of individuals suspected of abusing drugs. The hair concentration ranges, means, and medians are summarized for abused drugs in 158 authentic cases.     

New trends in hair analysis and scientific demands on validation and technical notes

This review focuses on basic aspects of method development and validation of hair testing procedures. Quality assurance is a major issue in drug testing in hair resulting in new recommendations, validation procedures and inter-laboratory comparisons. Furthermore recent trends in research concerning hair analysis are discussed, namely mechanisms of drug incorporation and retention, novel analytical procedures (especially ones using liquid chromatography-mass spectrometry (LC-MS) and alternative sample preparation techniques like solid-phase microextraction (SPME)), the determination of THC-COOH in hair samples, hair testing in drug-facilitated crimes, enantioselective hair testing procedures and the importance of hair analysis in clinical trials. Hair testing in analytical toxicology is still an area in need of further 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.     

应用HID Ion GeneStudioTM S5测序系统探讨毛干样本线粒体DNA异质性

目的应用HID Ion GeneStudio^TM S5测序系统对毛干样本线粒体全基因组分型结果的异质性进行探讨。方法采集8名无关个体的口腔拭子、血液及同一个体不同部位毛干样本,使用Precision ID mtDNA Whole Genome Panel对线粒体全基因组进行扩增,应用HID Ion GeneStudio^TM S5测序系统对线粒体全基因组进行分析检测。结果2名个体的颞部毛干样本线粒体DNA出现异质性,其余6名无关个体的口腔拭子、血液及不同部位毛干样本的线粒体全基因组分型结果均一致。8名无关个体共观察到119个碱基变异,个体的变异位点数目分别为29、40、38、35、13、36、40和35。结论应用HID Ion GeneStudio^TM S5测序系统可全面了解序列多态性。     

Shampoo residue profiles in human head hair

Washing hair with shampoo results in an accumulation of shampoo components in the hair. Hair of individuals using different shampoos can be distinguished by analysis of shampoo residues. A method for extraction and analysis of such residues is presented. The hair is extracted using a methanol/water mixture, and the extract is analyzed by reverse-phase high-pressure liquid chromatography (HPLC). The detector system consists of two ultraviolet (UV) detectors connected in series. The method is nondestructive to hair and is sensitive enough to be applied to a single hair 5 to 10 cm in length. Residues from hair balsams are analyzed by this technique as well. The use of this method in forensic science examination of human head hair is demonstrated.     

Testing human hair for cannabis

To validate information on cannabis use, we investigated human hair and pubic hair for cannabinoids (THC and THC-COOH) by gas chromatography/mass spectrometry. Samples (100 mg approximately) were decontaminated with methylene chloride, then pulverized and dissolved in 1 ml 1 N NaOH for 10 min at 95 °C in the presence of 200 ng of deuterated standards. After cooling, samples were extracted by n-hexane/ethyl acetate after acidification with acetic acid. After derivatization of the dry extract by PFPA/PFP-OH, the drugs were separated on a 30-m capillary column and detected using selected-ion monitoring (m/z 377 and 459 for THC and THC-COOH, respectively). Forty-three hair samples were obtained from fatal heroin overdose cases. Among them, 35% tested positive for cannabinoids. Hair concentrations ranged from 0.26 to 2.17 ng/mg (mean, 0.74 ng/mg) and 0.07 to 0.33 ng/mg (mean, 0.16 ng/mg) of THC and THC-COOH, respectively. As is generally the case for other drugs detected in hair, metabolite concentration was always lower when compared to the parent drug concentration. In pubic hair, THC concentrations ranged from 0.34 to 3.91 ng/mg (mean, 1.35 ng/mg) and THC-COOH concentrations from 0.07 to 0.83 ng/mg (mean, 0.28 ng/mg). In most cases, the highest cannabinoid concentration was found in pubic hair, suggesting that this sample may be the more suitable for cannabis testing.     

Evaluation and quantification of nuclear DNA from human telogen hairs

Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.     

Optimising direct PCR from anagen hair samples

Anagen hairs are in the active growth phase, and when forcefully removed, may contain an intact root or sheathing. The hair root or sheathing is a source of nucleic DNA and can be amplified using direct PCR. Human identification STR kits are optimised to a small range of input DNA for PCR. Anagen hairs are unable to be quantified prior to amplification and can exhibit characteristics of an over-loaded DNA sample when analysed. The aim of this study was to optimise direct PCR for anagen hair sampling. Two separate modifications to the downstream processes were carried out in order to determine the most effective method at minimising PCR artefacts. Decreasing the cycle number from the standard 29 cycles to 27 cycles when using the NGM™ kit displayed the best results for this method. However, decreasing the cycle number may increase allelic drop-out and would be costly for laboratories to perform an in-house validation. Diluting the PCR product during electrophoresis analysis minimises the effects of PCR artefacts in the same way decreasing the cycle number does. Diluting the PCR product is the most cost-effective method and does not increase the chance of allelic drop-out.     

Accumulation of explosives in hair--part II: factors affecting sorption

This study examines the sorption of eight explosives (2,4,6-trinitrotoluene [TNT]; pentaerythritol tetranitrate [PETN]; hexahydro-1,3,5-trinitro-s-triazine [RDX]; diacetone diperoxide [DADP]; triacetone triperoxide [TATP]; ethylene glycol [EGDN], nitroglycerin [NG]; and 2,4-dinitrotoluene [DNT]) to human hair. The study uses only cut hair, which is exposed to explosive vapor. The vapor transfer studies reported herein indicated that hair did not reach saturation even after 2.5 years of exposure to TNT. While previous studies showed black hair sorbed more explosive than blond or brown, this study reports that red hair sorption is similar to black, while grey hairs, exposed along with black hair from the same individual, sorbed significantly less explosive than the same individual's black hairs. In a study using only black hair, a slight racial bias was observed with sorption greater for Mongoloid hair as compared to Caucasian or Negroid. Only for Mongoloid hairs were enough samples studied to examine for a gender bias, but one was not observed. There was much variability in results in all categories (hair color, race, and gender) that trends were established only in general terms. Hair at different ages was tested for a few individuals. Detailed studies focused on the sorption of TATP and TNT as these appear to be sorbed most differently-TATP mainly on the hair surface and TNT both on the surface and in the cortex. The uptake of high vapor pressure explosives (e.g., TATP) and moderate vapor pressure explosives (e.g., TNT) by hair was rapid and could be detected within about 1 h of exposure. Both explosives were readily sorbed by pure melanin.     

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.     

Segmental Hair Analysis—Interpretation of the Time of Drug Intake in Two Patients Undergoing Drug Treatment

The present study involved segmental testing of hair in two clinical cases with known dosage histories. Hair analysis confirmed the first patient's exposure to the prescribed sertraline and citalopram for several months. Citalopram was generally distributed along the hair shaft in accordance with the drug ingestion period. By contrast, “false” positive results were observed for sertraline in distal hair segments, corresponding to a period of no sertraline exposure, which may indicate incorporation from sweat or sebum, which transport the drugs along the hair surface. The second patient received various drugs during her treatment for brain cancer. Metoclopramide, morphine, oxazepam, paracetamol, sumatriptan, tramadol, and zopiclone, which had been part of the therapy, were all detected in the proximal hair segment. The results of these two cases indicated that results—especially concerning the time of drug intake—must be interpreted with caution and allow for the possibility of incorporation from sweat or sebum.     

Characterization of Mitochondrial DNA Sequence Heteroplasmy in Blood Tissue and Hair as a Function of Hair Morphology*,†,‡

Abstract: This study characterizes mitochondrial DNA (mtDNA) sequence heteroplasmy in blood tissue and hair as a function of hair morphology. Bloodstains (127 individuals) and head hairs (128 individuals) were typed using the mtDNA LINEAR ARRAY? assay. A total of 1589 hairs were interpreted: 1478 (93%) were homoplasmic and 111 (7%) exhibited heteroplasmy at one or more positions. Seventy‐one percent (82/116) of individuals were homoplasmic, whereas 29% (34/116) exhibited heteroplasmy in at least one hair. The results demonstrate intra‐ and inter‐tissue differences in heteroplasmy within individuals. Sequence heteroplasmy among hairs from each individual varied from 0 to 90%; the frequency does not differ significantly with population group, cosmetic treatment, age, gender, medulla morphology, region of the scalp, hair growth phase, or, when comparing living and deceased donors. However, the results support a correlation between heteroplasmy and hair pigmentation; typically, lighter‐pigmented hairs exhibit a higher incidence of sequence heteroplasmy compared to darker hairs.     

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.