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.     

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.     

A contribution to the discussion of probabilities and human hair comparisons

The paper by Gaudette and Keeping on "An Attempt at Determining Probabilities in Human Scalp Hair Comparison" in the Journal of Forensic Sciences (Vol. 19, No. 3, July 1974, pp. 599-606) has provoked considerable controversy. This paper highlights two of the sources of the controversy and shows how the probability, 1/4500, quoted by Gaudette and Keeping should be treated with caution. The necessity of the use of a likelihood ratio statistic is described. It is suggested that the hair examination form resulting from the responses to the questionnaire recently distributed by the authors and also the discussions at Quantico (Proceedings of the International Symposium on Forensic Hair Comparisons, 25-27 June 1985, Quantico VA) should be used to facilitate the collection of the data which will be necessary to enable a likelihood ratio statistic to be estimated effectively.     

Evaluation of the addiction history of a dead woman after exhumation and sectional hair testing.

In Greece, sectional hair analysis, in addition to clinical examination, has been used as a valuable tool for the confirmation of a person's history of drug use. The present report concerns the toxicologic analysis of the exhumed remains and hair samples of an 18-year-old woman. Postmortem toxicologic analysis of blood and urine confirmed recent opiate and cannabis use and indicated that death was associated with heroin abuse. Several months later, the woman's family asked for exhumation and reexamination of the body, insisting that the cause of death was homicide. The investigating judge ordered exhumation and new medicolegal examination of the body. The investigation of the drug profile along the hair shaft was undertaken by analyzing hair sections 1 cm from the hair root for morphine, 6-monoacetylmorphine, heroin, and cannabinoids. The total lengths of the hair samples ranged from 8 to 11 cm. The total morphine levels in the hair sections corresponding to the 3-month period before death were significantly lower (1.5-2.85 ng/mg) than those of the 4- to 10-month period before death (7.4-14.8 ng/mg). An interpretation of these results may be occasional drug use (with considerable attenuation of use during the last 3 months before death). Decrease of tolerance to heroin caused by abstinence and relapse in use could have been the cause of death.     

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.     

Cannabinoid concentrations in hair from documented cannabis users

Fifty-three head hair specimens were collected from 38 males with a history of cannabis use documented by questionnaire, urinalysis and controlled, double blind administration of delta9-tetrahydrocannabinol (THC) in an institutional review board approved protocol. The subjects completed a questionnaire indicating daily cannabis use (N=18) or non-daily use, i.e. one to five cannabis cigarettes per week (N=20). Drug use was also documented by a positive cannabinoid urinalysis, a hair specimen was collected from each subject and they were admitted to a closed research unit. Additional hair specimens were collected following smoking of two 2.7% THC cigarettes (N=13) or multiple oral doses totaling 116 mg THC (N=2). Cannabinoid concentrations in all hair specimens were determined by ELISA and GCMSMS. Pre- and post-dose detection rates did not differ statistically, therefore, all 53 specimens were considered as one group for further comparisons. Nineteen specimens (36%) had no detectable THC or 11-nor-9-carboxy-THC (THCCOOH) at the GCMSMS limits of quantification (LOQ) of 1.0 and 0.1 pg/mg hair, respectively. Two specimens (3.8%) had measurable THC only, 14 (26%) THCCOOH only, and 18 (34%) both cannabinoids. Detection rates were significantly different (p<0.05, Fishers' exact test) between daily cannabis users (85%) and non-daily users (52%). There was no difference in detection rates between African-American and Caucasian subjects (p>0.3, Fisher's exact test). For specimens with detectable cannabinoids, concentrations ranged from 3.4 to >100 pg THC/mg and 0.10 to 7.3 pg THCCOOH/mg hair. THC and THCCOOH concentrations were positively correlated (r=0.38, p<0.01, Pearson's product moment correlation). Using an immunoassay cutoff concentration of 5 pg THC equiv./mg hair, 83% of specimens that screened positive were confirmed by GCMSMS at a cutoff concentration of 0.1 pg THCCOOH/mg hair.     

Site of discovery in the bath tub. Evaluation of a fatality after four years using postmortem roentgen diagnosis]

On the one hand the following case demonstrates the problems of finding a dead body in the bathroom and on the other hand the importance of postmortem x-ray examinations. The corpse of an 65-year-old woman was discovered in her bath tub. At the body's feet there was a hair dryer. Investigations concerning murder were negative, circumstances appeared to be unsuspicious. That was why a postmortem examination was not performed. -4 years later murder had to be considered. After manual strangling the deceased and the hair dryer were layed into the bath tub. The corpse was exhumed and taken to a forensic autopsy. It was found that the hyoid bone and the superior horn of the thyroid cartilage were both fractured on the right. Damages caused by the preparation itself were excluded by using serial roentgenographs. These observations in combination with histological findings and the results of further investigations convincingly supported the suspicion of strangling by hand.     

Determination of phosphoglucomutase phenotypes in hair bulbs

The research was to determine a simple method of phosphoglucomutase phenotype identification in hair bulb. The agarose technique and electrophoresis on cellulose-acetate foil methods were chosen because a small quantity of the maternal available for examination. It was found out that 1 or 2 bulbs are sufficient to identify the PGM1 features if the electrophoresis method is applied and if more bulbs are available, the PGM3 characteristic can also be identified. The modified technique was used for staining the phosphoglucomutase phenotypes.     

Development of forensic medical expertise of sexual conditions in men

The necessity of new methodological approaches in forensic medical examination of sexual male conditions are discussed basing on the analysis of questionnaire surveys of isolated groups of men and forensic medical examinations of male victims accused of sexual crimes. How to update expertise of sexual male conditions including investigations of anorectal and erectile dysfunctions in shown.     

Arsenic speciation of two specimens of Napoleon's hair

Since 1960, it has been demonstrated by various analytical procedures that high concentrations of arsenic were present in Napoleon's hair. Various authors, indicating that the detected arsenic levels are a consequence of external contamination, have challenged the results of these examinations. We have tested two strands of hair, referenced as Noverraz and Grand Maréchal Bertrand. Samples were incubated 6h in water at 90 degrees C, and arsenic speciation was carried out by HPLC-ICP/MS. The residue was injected on a cation-exchange PRP-X200 column that allows the detection of arsenobetaine and on an anion-exchange PRP-X100 column to test for the mineral forms. In these conditions, the inorganic species As(III), As(V) and their metabolites (DMA and MMA) were separated. Analysis of hair samples highlighted massive amounts of total arsenic (42.1 and 37.4ng/mg). Arsenical species found in the two samples of analyzed hair are distributed in the following: As(III) 31.1 and 44.7%; As(V) 66.3 and 53.2%; DMA 0.42 and 0.15%. Traces of MMA were detected, and 2% of the species could not be identified. These results prove that more than 97% of the arsenic found in the hair of Napoleon is in inorganic form, which is consistent with a chronic intoxication to arsenic.     

Practical GC/MS analysis of oxidation dye components in hair fiber as a forensic investigative procedure

The purpose of this study was to improve the reliability of discrimination (or identification) of dyed hair by analyzing chemical substances present in the hair, as an addition to the conventional macroscopical and microscopical examinations and ABO blood group examination. Oxidation hair-dye components such as p-phenylenediamine (PPDA), toluylene-2,5-diamine (T-2,5-DA), o-aminophenol (OAP), m-aminophenol (MAP), p-aminophenol (PAP) and p-amino-o-cresol (PAOC) were selected as the object of study. After alkaline-digestion, hair samples were adjusted to a pH of 12.6 to 12.8, and applied onto an Extrelut column. After 15 min, the components were simultaneously extracted and derivatized with n-hexane including 1% heptafluoro-n-butyryl (HFB) chloride. Their HFB derivatives within a condensed sample were diluted in ethyl acetate, and analyzed by gas chromatography-mass spectrometry (GC-MS) with full mass scanning or selected ion monitoring. For estimating their levels, 2,4,6-trimethylaniline was used as the internal standard. Standard curves obtained by preparing a 5 cm segment of control hair spiked with authentic substances were linear, ranging from 0.1 to 4.0 micrograms for PPDA and T-2,5-DA, and from 0.01 to 0.4 microgram for OAP, MAP, PAP and PAOC. The coefficient of variation of inter-day precisions (each n = 4) was below 16% for PPDA, below 20% for OAP and PAP and below 24% for T-2,5-DA, MAP and PAOC. These components were detectable even at 6 ng for PPDA, T-2,5-DA, MAP and PAP, 8 ng for OAP, and 4 ng for PAOC. Recovery percents using this procedure ranged from 54 to 86%. By using actual dyed hair samples this method was shown to provide high sensitivity for accurate detection.     

Identification of a forensic case using microscopy and forensically informative nucleotide sequencing (FINS): A case study of small Indian civet (Viverricula indica)

The exhibits obtained in wildlife offence cases quite often present a challenging situation for the forensic expert. The selection of proper approach for analysis is vital for a successful analysis. A generalised forensic analysis approach should proceed from the use of non-destructive techniques (morphological and microscopic examination) to partially destructive and finally destructive techniques (DNA analysis). The findings of non-destructive techniques may sometime be inconclusive but they definitely help in steering further forensic analysis in a proper direction. We describe a recent case where a very small dried skin piece (< 0.05 mg) with just one small trimmed guard hair (0.4 cm) on it was received for species identification. The single guard hair was examined microscopically to get an indication of the type of species. We also describe the extraction procedure with a lower amount of sample, using an automated extraction method (Qiagen Biorobot EZ1^®) and PCR amplification of three mitochondrial genes (16s rRNA, 12s rRNA and cytochrome b) for species identification. Microscopic examination of the single hair indicated a viverrid species but the initial DNA analysis with 16s rRNA (through NCBI BLAST) showed the highest homology (93%) with a hyaenid species (Hyaena hyaena). However, further DNA analysis based on 12s rRNA and cytochrome b gene proved that the species was indeed a viverrid i.e. Viverricula indica (small Indian civet). The highest homology shown with a Hyaenid species by the 16s rRNA sequence from the case sample was due to lack of a 16s rRNA sequence for Viverricula indica in the NCBI data base. The case highlights the importance of morphological and microscopic examinations in wildlife offence cases. With respect to DNA extraction technology we found that automatic extraction method of Biorobot EZ1^® (Qiagen) is quite useful with less amount of sample (much below recommended amount).     

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 and fingernail gadolinium ICP-MS contents in an overdose case associated with nephrogenic systemic fibrosis

Nephrogenic systemic fibrosis (NSF) is a rare acquired disorder affecting renal insufficiency patients. There is a growing recognition of the association between the rapid rise in the use of gadolinium (Gd)-containing contrast agents for magnetic resonance imaging (MRI), and the occurrence of acute nephrogenic systemic fibrosis. This case report concerns a 62-year-old woman with chronic renal insufficiency who underwent numerous MRI examinations using Gd-based contrast agents. For the first time, to our knowledge, Gd was performed in the NSF patient's blood, as well as in hair and in fingernails by inductively plasma coupled mass spectrometry. Gd contents in blood, hair and in fingernails were initially 300 to 1000 times higher compared to controls. These results are a new illustration of the strong association between the frequent use of Gd-based contrast media and NSF in patients with kidney diseases. Gd quantitative determination could be of major interest for patients with renal failure who are required to undergo repeated gadolinium-enhanced MRI examinations.     

Arsenic speciation of two specimens of Napoleon's hair

Since 1960, it has been demonstrated by various analytical procedures that high concentrations of arsenic were present in Napoleon's hair. Various authors, indicating that the detected arsenic levels are a consequence of external contamination, have challenged the results of these examinations. We have tested two strands of hair, referenced as Noverraz and Grand Maréchal Bertrand. Samples were incubated 6 h in water at 90 °C, and arsenic speciation was carried out by HPLC–ICP/MS. The residue was injected on a cation-exchange PRP-X200 column that allows the detection of arsenobetaine and on an anion-exchange PRP-X100 column to test for the mineral forms. In these conditions, the inorganic species As(III), As(V) and their metabolites (DMA and MMA) were separated. Analysis of hair samples highlighted massive amounts of total arsenic (42.1 and 37.4 ng/mg). Arsenical species found in the two samples of analyzed hair are distributed in the following: As(III) 31.1 and 44.7%; As(V) 66.3 and 53.2%; DMA 0.42 and 0.15%. Traces of MMA were detected, and 2% of the species could not be identified. These results prove that more than 97% of the arsenic found in the hair of Napoleon is in inorganic form, which is consistent with a chronic intoxication to arsenic.     

Hair and urine testing to assess drugs of abuse consumption in couples undergoing assisted reproductive technology (ART)

For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated. Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples. Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers.     

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.     

Subgaleal hematoma in child abuse

The mother brought her 31/2-year-old son to a paediatric clinic as his forehead was swollen and bluish in appearance. She was unable to give any explanation for this lesion. Clinically it was noted that a very visible and also palpable, doughy swelling was present both in the forehead as well as the hard skull areas. Some hours later, a massive bilateral periocular haematoma appeared. Whilst X-rays were unable to provide any diagnostic help, the sonographic examination of the skull carried out on the same day revealed an extensive subgaleal haematoma. This phenomenon is described in radiological literature as "scalping" and is caused by the use of considerable, blunt violence against the head (shear stresses, e.g. by pulling at the hair). The mother's boyfriend later confessed forceful pulling of the hair.     

Possibilities of DNA sex determination in hair roots

Species- and sex-determination on hair roots were simultaneously performed using extracted DNA and a human Y chromosomespecific probe. (pH Y 2.1) After Southern hybridization, Hae III-digested DNA fragments were detected by non-radioactive digoxigenin detection system. DNA was extracted from one to five freshly plucked hair roots. The specific 2.12 kb fragment was successfully detected in male DNA samples from a single hair root. A positive identification of female DNA was more difficult. The hair root DNA was revealed to be stable at room temperature for at least 2 weeks (examination time) and produced the same specific band pattern as the DNA of fresh hair roots. In the blind tests with DNA samples from randomly plucked one to four hair roots, the rate of successful sex-determination was 95.8% on male samples (23 out of 24 samples) and 25% on female samples (4 out of 16 samples).