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.     

Determination of ABO blood group markers in single hair segments

Determination of ABO-blood group on human hair segments of 79 individuals was performed by means of modified Yada absorption-elutions technique. The studies were carried out with 4 micron thin cross sections of hair. In 79% of the cases it was possible to establish the correct blood group. Individual hair segments of 0.5 cm length yielded reliable results. Antigen identification was best with blood group B and most difficult of the AB type.     

Experimental statistical studies on the evaluation of the value of evidence in the comparison of human hair

In this paper a statistical experiment is presented which allows to estimate the evidential value of human head hair comparison. The procedure described in detail was essentially the same as in case-work. Questioned hairs were taken randomly from 20 different persons out of a pool of 111 individuals. From each of the 20 persons one, three an five hairs respectively were compared consectively with samples from 100 different individuals. The results were classified as follows: a) "matching" and b) "similar" - if the hair(s) may originate form that person; c) "not matching" - if an individual is excluded as possible source. In our experiment about 95% of the samples (persons) were excluded as possible source of a questioned hair on the average. This is a mean value which may vary considerably in a distinct case. The experiment, its results and problems are discussed.     

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.     

DIA3 phenotyping in human tissues, dental pulps, and hair roots by isoelectric focusing

The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.     

Use of the isoelectric focusing method in examining hair proteins

Method of isoelectric focusing in thin and ultrathin layer of polyacrylamide gel followed by staining the polypeptide fractions with argentum was used to investigate human hair keratins. Method makes it possible to determine the polymorphism of keratins in hair even 1,5-2 cm long. It helps to obtain the additional information in medicolegal expertise of hair affinity.     

Sexual abuse and anti-wrinkle cream: evidence from octocrylene

In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC-MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter.     

USGS42 and USGS43: human-hair stable hydrogen and oxygen isotopic reference materials and analytical methods for forensic science and implications for published measurement results

Because there are no internationally distributed stable hydrogen and oxygen isotopic reference materials of human hair, the U.S. Geological Survey (USGS) has prepared two such materials, USGS42 and USGS43. These reference materials span values commonly encountered in human hair stable isotope analysis and are isotopically homogeneous at sample sizes larger than 0.2 mg. USGS42 and USGS43 human-hair isotopic reference materials are intended for calibration of δ(2)H and δ(18)O measurements of unknown human hair by quantifying (1) drift with time, (2) mass-dependent isotopic fractionation, and (3) isotope-ratio-scale contraction. While they are intended for measurements of the stable isotopes of hydrogen and oxygen, they also are suitable for measurements of the stable isotopes of carbon, nitrogen, and sulfur in human and mammalian hair. Preliminary isotopic compositions of the non-exchangeable fractions of these materials are USGS42(Tibetan hair)δ(2)H(VSMOW-SLAP) = -78.5 ± 2.3‰ (n = 62) and δ(18)O(VSMOW-SLAP) = +8.56 ± 0.10‰ (n = 18) USGS42(Indian hair)δ(2)H(VSMOW-SLAP) = -50.3 ± 2.8‰ (n = 64) and δ(18)O(VSMOW-SLAP) = +14.11 ± 0.10‰ (n = 18). Using recommended analytical protocols presented herein for δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurements, the least squares fit regression of 11 human hair reference materials is δ(2)H(VSMOW-SLAP) = 6.085δ(2)O(VSMOW-SLAP) - 136.0‰ with an R-square value of 0.95. The δ(2)H difference between the calibrated results of human hair in this investigation and a commonly accepted human-hair relationship is a remarkable 34‰. It is critical that readers pay attention to the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) of isotopic reference materials in publications, and they need to adjust the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurement results of human hair in previous publications, as needed, to ensure all results on are on the same scales.     

汉人不同区段头发的线粒体HVII异质性的序列分析

目的探讨汉族人不同区段头发线粒体DNA(mtDNA)HVII区的异质性。方法用5%Chelex100法提取7名汉族个体额、顶、枕及左、右颞部等5个不同部位的不同根不同段的头发mtDNA,同时取各自毛囊作为对照;以两步法扩增纯化后测序反应,3100型遗传分析仪检测。结果不同毛干区段的点异质性多发生于女性长发远段、儿童及老年人,不同区及同一根不同段均可发生点异质性,可多达4处,点异质性可能相同,可能不同,但一般多发生于相同个体毛囊mtDNA点突变处。不同区头发长度异质性不同,同一根头发不同段长度异质性相同。mtDNA点异质性有一定遗传倾向。稀释及混合样本mtDNA图谱也可表现为“点异质性”图谱。结论根据人头发毛干mtDNA测序结果得出“排除”结论时一定应慎重。     

Hair analysis for fenfluramine and norfenfluramine as biomarkers for N-nitrosofenfluramine ingestion

In this paper, a high performance liquid chromatographic method with fluorescence detection (HPLC-FL) for the determination of fenfluramine (Fen) and norfenfluramine (Norf) in human hair as biomarker metabolites of N-nitrosofenfluramine (N-Fen) is described. Washed and cut hair segments were extracted by ultrasonication for 1h at room temperature in methanol. The extract was evaporated and applied for derivatization with the fluorescent reagent 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). An HPLC-FL analysis was performed using an ODS column with mobile phase composition of acetonitrile and water (65:35, v/v) and monitored at 430 nm (excitation 325 nm). The method was sensitive with detection limits of 36 and 16 pg/mg hair for Fen and Norf, respectively. The linearity was assessed in the range 0.036-144 ng/mg for Fen and 0.016-127 ng/mg for Norf with correlation coefficients larger than 0.999. The method was successfully used for the segmental determination of Fen and Norf in hair samples obtained from hospitalized patients diagnosed with hepatotoxicity and suspected to ingest N-Fen. Both Fen and Norf could be detected in these patients' hair samples in the ranges 43-1389 pg/mg for Fen and 18-680 pg/mg for Norf and the results showed that the patients might ingest N-Fen for a period of not less than 5 months. As well, the method was applied for the determination of Fen and Norf in rats that possess pigmented and non-pigmented hair after an intraperitoneal administration of Fen. Both compounds were determined in black as well as in white hair.     

微波消解ICP-MS法测定人发中的汞

目的建立人发中汞的电感耦合等离子体质谱分析方法。方法采用微波消解法处理样品,以铟（115In）作内标,用电感耦合等离子体质谱分析人发中的汞含量。结果方法检出限为0.0032μg/g,准确度通过测定人发标准物质GBW07601、GBW09101b进行验证,检测结果与标准参考值相符。结论该方法快捷、高效,灵敏度、准确度高,适用于人发中汞含量的检测。     

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 detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.     

人发毛干角蛋白电泳谱型的分析

本文用SDS-梯度(5.0～17.0％)聚丙烯酰胺凝胶电泳对150例中国人头发角蛋白组分进行了分析。结果表明,在150例人头发中,有6种不同类型的角蛋白电泳谱型。此1～6型发生频率分别为24％、12％、42％、7.3％、8.0％;及6.7％。用N-(3-芘)马来酰胺标记头发角蛋白巯基,证实人头发不同类型的角蛋白电泳谱型主要区别在低硫蛋白部位。作者认为,人头发角蛋白电泳谱型的差异可为法医学鉴定中的毛发个人识别提供重要的依据。     

Detection of meperidine and its metabolites in the hair of meperidine addicts

The presence of meperidine and its metabolites in the hair of meperidine addicts was investigated using GC–MS (EI, PCI). Meperidine and its three metabolites – normeperidine, N-methoxy meperidine and acetyl normeperidine, were found in hair samples from addicted subjects. Methods for the simultaneous determination of meperidine and its metabolites by GC–MS-SIM were also established for human hair samples. After the addition of d₄-meperidine as an internal standard, hair samples weighing 5 mg were incubated in 0.1 M HCl at 45°C overnight, and the resulting digests were extracted with ether. The recoveries were greater than 80%, with coefficients of variation (CVs) between 4.48 and 8.31%. The calibration curves for meperidine and normeperidine in hair were linear over a concentration range of 1 to 500 ng per mg of hair, with correlation coefficients of r=0.9990 and r=0.9992, respectively. Values less than 0.25 ng/mg of hair were cut off. Hair samples obtained from 60 drug addicts were analyzed using this method, and the content of meperidine and normeperidine was determined to be 103±130 and 117±143 ng/mg, respectively. Sectional analysis revealed that meperidine was present and stable in hair for at least 20 months, but normeperidine content at the level of the hair root was higher compared to the tip of the hair shaft. The results also revealed that there was a correlation between the subject’s drug abuse history and the distribution of drug along the hair shaft, and between the doses of meperidine and drug content presented in hair.     

氯胺酮滥用的毛发分析研究

目的建立毛发中氯胺酮及其代谢物的分析方法并探索氯胺酮进入毛发的机理。方法通过建立豚鼠连续给药(不同剂量)实验模型获取阳性头发和采集氯胺酮滥用者头发,经处理后用GC/MSscan和SIM法分析,以鉴别、确认毛发中氯胺酮及其代谢物。结果豚鼠毛发中氯胺酮的质量分数与给药剂量存在明显的正相关性。毛发中氯胺酮质量分数依白色、棕色、黑色毛发顺序随毛发中黑色素的质量分数增加而增加。豚鼠毛发中氯胺酮与代谢物NK质量分数之比为2.33～12.94,仅在高剂量组的豚鼠毛发中才检测到DHNK,其质量分数与NK接近。15名氯胺酮滥用者黑色头发中均检出原体和代谢物NK,但DHNK少见。豚鼠毛发中代谢物相对质量分数明显高于人。结论本实验结果很好地反映了药物进入毛发代谢过程与药物和黑色素亲和力以及药物的亲脂性密切相关这一规律,但人和动物在药物代谢及进入毛发的难易程度上存在差异。本方法可以用于法庭毒物分析领域头发中氯胺酮的检测。     

Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy

The increasing interest in toxicological hair analysis as a marker of human exposure to xenobiotics such as illicit substances or therapeutic drugs, has been made feasible by the extension of mass spectrometry, a highly sensitive method of detection. A newborn exposed to drugs in utero can suffer from a varying degree of withdrawal syndrome, a few days after birth. If of opiate origin, the withdrawal syndrome can be treated with morphine, among other therapeutics, but it is not easy to diagnose because of atypical symptoms presented by neonates and especially when maternal drug addiction has not been revealed. To assess and measure toxicological factors linked with the appearance and the severity of this syndrome, maternal and neonatal matrices such as urine, meconium and hair were collected during a protocol approved by the ethical committee. Opiates in particular were measured with GC-MS and potential combined dependences (cannabis, cocaine, amphetamine, LSD and benzodiazepines) and/or substitutive therapeutics (methadone or buprenorphine) were also assessed in 17 mother/neonate couples. Gestational opiate exposure profiles were drawn up and linked with the observed withdrawal syndromes. A withdrawal syndrome seems to appear more frequently after foetal exposure to an association of opiates/substitutive molecules (8 out of 10 withdrawal syndromes observed in this study), although the impact of cocaine and benzodiazepines must also be taken into account. The results obtained in neonatal hair make it possible to affirm foetal drug exposure and are in accordance, for the majority, with the appearance of a neonatal withdrawal syndrome (NWS). Neonatal hair analysis could contribute to assess in utero exposure to opiates, particularly when results in urine and meconium are negative or when these matrices are not available.     

Interlaboratory comparison of quantitative determinations of drug in hair samples

Testing human hair for drugs of abuse is a relatively new technique which requires control before being fully accepted in Justice applications. A consensus procedure was recently proposed to the four French Laboratories performing hair analysis for opiates and cocaine. Results of two independent controls have shown that the laboratories have performed very well quantitatively, using the recommended method. In order to compare these results with those obtained by other procedures, one sample was sent to 15 laboratories concerned with the analysis of human hair for drugs of abuse in Germany, Italy, Spain, and United States. Results from this study have indicated that the French recommended method is in accordance with the general procedures.     

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.     

The use of supercritical fluid extraction for the determination of amphetamines in hair

A laboratory study interested in the analysis of human hair for drugs-of-abuse was conducted to determine if drugs could be detected and quantified from hair. Supercritical fluid extraction (SFE) techniques followed by GC-MS analysis were applied to extract amphetamines from hair. The group of amphetamines included methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and internal standard mephentermine (MP). To validate information on amphetamine use in hair, powdered hair samples free from drugs were collected and soaked in a known amphetamine standard solution. Authentic fortified case hair samples taken from known drug users known to have consumed amphetamines were also analyzed for amphetamine. Results from this study show that amphetamine use can be detected in spiked and authentic fortified human hair using SFE techniques for qualitative and quantitative reproducible results.