Degeneration of Nuclei and Mitochondria in Human Hairs

Abstract:  It is generally accepted that nuclei degrade in developing hair shafts but the point at which such occurs has not been investigated. The fate of mitochondria in the keratinizing hair shaft has been less clear. This study uses transmission electron microscopy to investigate when nuclei and mitochondria are no longer visible in the developing hair shaft. Serial sections were obtained from anagen head hairs absent follicles in order to determine the sequence of degradation of nuclei and mitochondria in the hair shaft by starting at the root bulb and proceeding toward the hair tip. It was demonstrated that nuclei and mitochondria become invisible in the keratinizing hair shaft at about the same time. This was found to occur fairly early in the process at the level of the hair shaft where the hair cuticle becomes permanent.     

Theoretical limits of the evaluation of drug concentrations in hair due to irregular hair growth

When examining concentration relationships of doses it must be taken into account that hair growth is irregular. Hair growing from the shaved skin after a single dose of a certain drug cannot possibly contain the same concentration as hair after the same dose that has not been cut over a long period. Concentrations can even change during the hair growth in cases where the hair had been cut a couple of months before the hair sample was taken. The variations in the expected concentrations can exceed 20%. On the other hand, the evaluation of a hair tuft which has grown after the last drug consumption may be important in forensic cases where the hair which has grown earlier is not available. This may lead to misinterpretations at low concentrations. Expected concentrations are calculated assuming a telogen part of 10%.     

The role of variations in growth rate and sample collection on interpreting results of segmental analyses of hair

Segmental analysis of hair for drugs, metabolites, and poisons has been widely reported in the scientific literature over the past two decades. Two fundamental assumptions in interpreting results of such analyses are (1) an average linear growth rate of head hair of 1cm/month and (2) that sample collections occur with the hair being cut directly next to the scalp. The purpose of this study was to evaluate the variability associated with growth rate of human head hair, as well as the ability to uniformly collect hair next to the scalp. The results were used to determine how these factors affect the interpretation of results generated in segmental analysis of hair. A thorough literature review was conducted to assess the range of linear growth of human head hair from the vertex posterior and occipital regions. The results were compiled to establish the average (1.06cm/month), as well as the range of possible growth rates of head hair. The range was remarkable and suggests that conclusions based on the 1-cm/month growth rate could be significantly skewed. A separate study was undertaken to evaluate collection of hair next to the scalp. Fourteen individuals were provided oral instructions, as well as a written standard collection procedure for head hair. The experience levels among the collectors varied from novice to expert. Each individual collected hair from dolls with short- and long-hair. Immediately following each collection, the sampling area was evaluated to determine how close to the scalp the cuts were made, as well as the variability in the lengths of hair remaining at the sampled area. From our collection study, we determined that 0.8±0.1cm of hair was left on the scalp after cutting. When taking into account the amount of hair left on the scalp after collecting, the use of a growth rate of 1.06cm/month, and the assumption that it takes two weeks for newly formed hair in the follicle to reach the scalp, we find that the first 1-cm segment of hair typically corresponds to hair formed 1.3±0.2 to 2.2±0.4 months (95% confidence) earlier. The impact of these findings as it relates to the corresponding time for each additional segment is demonstrated. As a result, we recommend that hair collection be delayed 8 weeks after a suspected ingestion to ensure that the sample fully represents the exposure period. The results of this study suggest that the variability in the growth rate of human head hair, as well as the inconsistent collection of hair, significantly affect the interpretation of results from segmental analysis of hair.     

Examinations of ABO bloodgroupings of human hair

In order to bring into play the function of human hair ABO bloodgrouping in the field of medicolegal expertise on material evidence and raise its accuracy, the author, through a test on human hair of 500, makes some emphatic researches on the disposal of hairs, on the application of anti-A and anti-B serum, on the selection of red blood cell indicator and on the elution temperature as well. Five hundred samples of human hair have been examined upon the basis of the improved operation method and through the application of anti-A and anti-B serum, the titer of which being 1:128, and therein, fine results have been obtained.     

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.     

Decontamination procedures for drugs of abuse in hair: are they sufficient?

This paper reviews the methods for decontaminating hair exposed to external solutions of drugs of abuse. Exposure of hair to cocaine at 1 μg/ml for 5 min is sufficient to contaminate hair, yet decontamination is a very slow process. Using externally contaminated hair, a number of decontamination procedures were attempted, and none removed all the contamination. The percentage of external contamination removed depended on the hair type, with thick black hair being the most resistant to decontamination. Hair treated by dying incorporated externally applied drugs differently, depending on the hair type. Thick black hair became more absorbent whereas thin brown hair became less absorbent. Kinetic wash criteria are evaluated for their ability/inability to determine if hair has been contaminated from external sources. A theoretical framework for the incorporation and removal of drugs from hair is discussed, and the hypothesis that inaccessible domains exist in hair which trap drugs is critically examined. The results presented in this paper strongly suggest that much more information on the decontamination of hair and the differentiation of exogenously and endogenously incorporated drugs is needed before hair analysis can be employed in most forensic applications. We propose that the radioactive tracer methods discussed herein are well suited for evaluating any new decontamination or extraction technique.     

Sex determination from plucked human hairs without epithelial root sheath

For sex determination, it is difficult to examine the sex chromatin in the hair root devoid of the epithelial root sheath because of melanin deposition in the hair cortex cells. In the present study, the possibility of determining sex was sought by observing the frequency of Y chromatin in the nuclei of hair cortex cells from human hair root devoid of epithelial root sheath. The male hair contained nuclei in each of which one brightly fluorescent spot was recognizable. The average frequency of the fluorescent spot in the hair cortex nuclei observed in 90 samples from 30 males was 59.4%; the maximum frequency was 74% and the minimum 37%. On the other hand, the fluorescent spots were rarely found in the nuclei of hair cortex cells from female hair. The frequency of occurrence was 5.3% on average, the maximum frequency being 12%, the minimum 0%. The frequency distribution of the male group and female group exhibited two discontinuous peaks. The frequency of the fluorescent spots can therefore be used as a criterion for determining sex. The fluorescent spot in dried male hair was shown to be detectable in a frequency of 20 – 40% 4 weeks after plucking, indicating that the nuclei of hair cortex cells can be subjected to the examination for at least this period if the hair has been kept in a dry condition.     

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.     

Do drug users use less alcohol than non-drug users? A comparison of ethyl glucuronide concentrations in hair between the two groups in medico-legal cases

Two groups were selected from the remainder of hair samples that had been tested for drugs at TrichoTech for medico-legal cases: samples that tested negative (drug-negative group; N=42, age 33.4+/-7.2 years) and samples that tested positive for drugs (drug-positive group; N=57, age 32.5+/-8.8 years). A rapid, simple method to detect the ethanol metabolite, ethyl glucuronide (EtG) in hair has been developed. The hair samples were sectioned, and then submitted to overnight sonication in water. Samples then underwent SPE using anion exchange cartridges, followed by derivatisation with N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA), before confirmation by GC-MS/MS. The assay produced excellent linearity and sensitivity over the calibration range 0.02-1.0 ng/mg, assuming a 10 mg hair sample. The mean age of the two groups was not statistically different (p=0.575, Student t-test), indicating a homogeneous group. Twelve of the 57 (21.0%) hair samples of the drug-positive group tested positive for EtG, and 17 of the 42 (40.5%) hair samples of the drug-negative group tested positive for EtG. The mean concentration of EtG in the drug-positive group was 0.011 ng/mg compared to 0.107 ng/mg in the drug-negative group. When the full results of this study were subjected to statistical analysis it was shown that EtG levels in the drug-negative group were statistically higher than those found in the drug-positive group (p<0.05). This preliminary finding may be of use in the study of addiction and adds valuable data to previous studies regarding the use of EtG as a valuable marker for alcohol levels in hair.     

Preliminary investigation into the recovery of explosives from hair.

Sampling of hair has proved to be a useful non-invasive method for detecting illicit drugs. This study examined the viability of hair as a surface from which explosive traces can be recovered and showed that as little as one-hour vapour exposure can result in measurable traces of explosives. Contamination of the hair may result from direct contact with explosive particles or from secondary contact by hand. Also the paper demonstrates that hair can concentrate explosive from the ambient vapour of a variety of military explosives. It was found that the amount of TNT picked up by the hair increased with time of vapour exposure. The data also suggested that unwashed hair may pick up more TNT than washed hair.     

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.     

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.     

Removing and identifying drug contamination in the analysis of human hair

The procedure used in this laboratory for removing and identifying contamination of hair specimens with drugs is demonstrated by its application to hair contaminated by various experimental models. The models include soaking; coating with drug followed by sweat conditions for 6 h; and soaking in a very high concentration of cocaine followed by storage and multiple shampoo treatments. A multi-part wash procedure along with a wash criterion is applied to all samples containing drug above the cutoff. The failure of the wash criterion is a signal that the sample may be positive due to contamination rather than use, and in the absence of other over-riding evidence, the sample would be considered to be negative for drug use. This Wash Criterion has also been tested with hair from subjects demonstrated to be drug users by one or more drug-positive urines; in these studies, all hair samples from demonstrated users passed the Wash Criterion test.     

Drug testing by urine and hair analysis: complementary features and scientific issues

Hair analysis and urinalysis are complementary tests for establishing drug use. Hair analysis provides long-term information, from months to years, concerning both the severity and pattern of drug use. In contrast to this, urinalysis can indicate only drug use, and then generally only that which has occurred within the last 2–3 days. Field studies have demonstrated that hair analysis is considerably more effective than urinalysis at identifying drug users. This difference is due to the wider surveillance window of hair analysis and to the susceptibility of urinalysis to evasive maneuvers. The main concerns with urinalysis are endogenous evidentiary false positives caused by passive drug exposure, e.g., ingestion of poppy seed. This problem arises from the hypersensitivity of the urine test, i.e. the need to use low cut-off levels in order to compensate for the temporary recording of drug use. This problem does not occur with hair analysis since its wide window of detection and permanent record of drug use ensure that the detection efficiency of the test is not compromised by the use of more effective cut-off levels guarding against passive endogenous drug exposure. On the other hand, exogenous evidentiary false positives due to external contamination of hair by drugs present in the environment (e.g., smoke) are the main concern of hair analysis. This problem, however, can be effectively avoided by washing the hair specimen, by kinetic analyses of the wash data, and by measurement of metabolites. The possibility of bias due to race and/or hair color is avoided by the exclusion of melanin from the analysis of hair. The safety and effectiveness of hair testing has been established by extensive field studies with over 400 000 specimens.     

Hair as a specimen to document tetramethylene disulfotetramine exposure

Tetramethylene disulfotetramine (tetramine) is a rodenticide that has been banned for many years in China. Since 2005, inhabitants of a village in the Henan Province have been suffering from grand mal seizures. To investigate the possibility of tetramine as the cause, we developed a method to determine tetramine in human hair. Sample preparation involved external decontamination, frozen pulverization, and ultrasonication in 2 mL ethyl acetate in the presence of cocaine-d3 as an internal standard. The method exhibited good linearity; calibration curve was linear over a range of 0.1-20 ng/mg hair. The limit of detection for the assay was 0.05 ng/mg hair. Except for one subject (No. 4), all head and pubic hair samples were positive for tetramine. The concentrations of tetramine in pubic hair were significantly higher than those in the same subjects' head hair samples. Because of a long retention in body, segmental head hair analysis cannot provide an accurate exposure history of tetramine in the body.     

Methadone and EDDP in hair from human subjects following a maintenance program: results of a pilot study

A specific method has been developed for the quantitative determination of methadone (MTD) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in hair.An amount of 50mg hair samples were incubated in 0.01M HCl overnight at 60 degrees C and deuterated internal standards of MTD and EDDP were added before extraction. Hydrolyzed solutions were extracted by automated solid-phase extraction procedure and analyzed on a gas chromatography (GC) coupled to a ion trap mass spectrometer (MS). Positive chemical ionization was used with acetonitrile as liquid reagent. The different validation parameters, linearity, repeatability, recovery and detection limits are presented. A relative standard deviation (R.S.D.) of 12 and 11% was obtained for the repeatability of MTD and EDDP, respectively. The limits of quantification (LOQ) was 0.05ng/mg for MTD and 0.2ng/mg for EDDP.A number of 26 hair samples from human subjects following a long-term MTD therapy were analyzed by this method. Blood samples of these subjects were analyzed with a routine method using a liquid-liquid extraction and GC/nitrogen phosphorus detector (NPD). MTD was quantified in blood and hair samples and EDDP found in 50% of the hair sample.A comparison was made between the concentrations found in blood or in hair and the dose administrated. This study could demonstrate that there is no relation between the administrated dose and MTD or EDDP concentrations in hair.     

The secondary transfer of fibres from head hair.

In this study, the effects of fibre type, hair style, time and fibre persistence on the secondary transfer of mask fibres to pillowcases via head hair were studied. Volunteers with a range of hair styles, and masks consisting of different fibre compositions were used in the study. Fibres from the masks were found to transfer from donor subjects to the pillowcases up to 14 nights after the mask had been worn. On average, the number of secondarily transferred fibres found decreased with time; however, this decrease appeared to be more 'linear' in nature, rather than an exponential decay. The greatest degree of secondary transfer occurred with cotton, then acrylic, then wool. In a primary transfer/persistence experiment with a 50% acrylic/50% wool mask, wool was found to persist in the hair more readily than acrylic. The results also showed that the greatest degree of secondary transfer occurred via short straight and long straight hair, with no clear pattern emerging between medium length hair (both straight and curly) and with long curly hair. The implications of these findings for the assessment and interpretation of casework are considered along with data obtained from related studies.     

Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.     

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.     

Analysis of drugs of abuse in hair: evaluation of the immunochemical method VMA-T vs. LC-MS/MS or GC-MS

Hair analysis is an elaborate and time-consuming multi-step process. The immunometric test VMA-T from Comedical has been evaluated as screening assay for hair analysis. From routine work, authentic samples were selected that were positive for opiates, cocaine, MDMA-type drugs, amphetamines, methadone or THC. These hair samples were investigated by LC-MS or GC-MS and the VMA-T procedure, respectively. Using the cut-off values recommended by the Society of Hair Testing, the VMA-T method discriminates with good sensitivity between negative and positive hair samples and is an expedient screening method for drugs in keratinized matrices such as hair. In order to save time and resources, the residue of the VMA-T extraction solution can be reused for confirmation analysis by LC-MS except for cocaine.