Single Hair Research Articles

Transplantation of autologous single hair units heals chronic wounds in autosomal recessive dystrophic epidermolysis bullosa: A proof-of-concept study

Autosomal recessive dystrophic epidermolysis bullosa (RDEB) is characterized by recurrent mucocutaneous blistering with non-healing ulcers which are often complicated by squamous cell carcinoma (SCC). Despite having as high as 80% death rate from SCC, RDEB still does not have an effective treatment. We report on the efficacy of single follicular unit extract (FUE) grafting to heal chronic ulcers of intermediate RDEB in a 54-year-old woman with extensive chronic wounds covering around 30% of the body surface area. On Day 17 post first graft session, the area of treated ulcers on her right upper back was reduced by 80%. Immunofluorescence study revealed positive type VII collagen expression along the epidermal and follicular basement membrane zone in the donor and recipient sites. A few grafted follicles continued to grow hair on the recipient sites. A total of 360 FUEs were grafted in nine sessions over five years, resulting in healing of most treated ulcers and reduced significantly her time for daily wound dressing. Importantly, FUE grafting using patient's own scalp follicles does not require any laboratory manipulation. It is safe and easy to perform. Autologous follicular grafting appears efficacious for healing of recalcitrant wounds and provides an innovative solution for RDEB patients with such wounds.

Structural elasticity for tensile deformation of a single human hair and the comparison with it for the bending deformation

Human hair is a multi-layered structure, which consists of the inner medulla, middle cortex, and outer cuticle. Therefore, the mechanical properties of the hair are related not only to the Young's modulus of each layer but also to the internal structures. Although the tensile test of a human hair has been performed elsewhere, the deformability of the hair for the tensile deformation is determined as the Young's modulus of the hair structure, which is similar to that of metals. In this paper, the structural elasticity of a single human hair for the tensile deformation, which expresses the deformability of a hair by tension without being dependent on external dimensions and shape, is defined based on the theoretical model, and is measured by performing the tensile test under the digital microscope observation. The values of the structural elasticity for the tensile deformation of the hair samples collected from healthy persons are compared with the values obtained for bending deformation. The structural elasticity for the tensile deformation of the hair sample is found to be lesser than that of the bending deformation, and this is verified to be always valid provided the Young's modulus of the outer cuticle is greater than that of the middle cortex.

Incorporation of Methoxyphenamine into Hair in Early Stage after Intake.

In order to investigate the incorporation behavior of drugs into hair in early stage (within 24 h) after intake, time-course changes in drug distribution in black hair were carefully analyzed after a single oral administration of methoxyphenamine (MOP), a non-regulated analog of methamphetamine. Single-hair specimens collected by plucking with the roots intact at appropriate intervals post-intake were each divided into 1-mm segments from the proximal end, and MOP in each segment was determined by a validated liquid chromatography-tandem mass spectrometry procedure. At 10 min after intake, MOP was not detected in any of the segments. MOP became detectable 30 min after intake in the hair bulb (0-1-mm segment from the proximal end) and 1 h after intake in the upper dermis zone (1-2-mm to 4-5-mm segments). The amount of MOP in the hair bulb increased rapidly over 3 h after intake and reached a maximum concentration of ∼100-900 pg/1-mm single hair (11-95 ng/mg) around 3-10 h after intake, whereas that in the upper dermis zone increased at a more gradual pace over 24 h and reached a plateau at ∼30-100 pg/1-mm hair (3-11 ng/mg). These differences can be attributed to the different incorporation mechanisms of the drug. Results from this study can further elucidate the drug incorporation mechanism, which is crucial for accurately interpreting results in hair analyses. Our findings also suggest that hair drug analysis with special attention to the hair root can serve as a useful complementary approach to urine- and blood-based testing in the field of forensic toxicology.

Detection of single human hairs with a terahertz nonlinear quantum cascade laser.

We report the demonstration of imaging of a single human hair with a terahertz quantum cascade laser (THz-QCL) source based on intracavity difference-frequency generation. A single human hair whose diameter was about 100 µm was detected using the THz-QCL source operating at 240 K, of which the THz beam had a linear polarization. The results show that the THz image of a human hair clearly depends on the polarization direction of the THz beam. The THz QCL sources that are capable of room temperature operation will be useful for detection of small foreign objects like human hairs.

Synaptojanin2 Mutation Causes Progressive High-frequency Hearing Loss in Mice.

Progressive hearing loss is very common in the human population but we know little about the underlying molecular mechanisms. Synaptojanin2 (Synj2) has been reported to be involved, as a mouse mutation led to a progressive increase in auditory thresholds with age. Synaptojanin2 is a phosphatidylinositol (PI) phosphatase that removes the five-position phosphates from phosphoinositides, such as PIP2 and PIP3, and is a key enzyme in clathrin-mediated endocytosis. To investigate the mechanisms underlying progressive hearing loss, we have studied a different mutation of mouse Synj2 to look for any evidence of involvement of vesicle trafficking particularly affecting the synapses of sensory hair cells. Auditory brainstem responses (ABR) developed normally at first but started to decline between 3 and 4 weeks of age in Synj2tm1b mutants. At 6 weeks old, some evidence of outer hair cell (OHC) stereocilia fusion and degeneration was observed, but this was only seen in the extreme basal turn so cannot explain the raised ABR thresholds that correspond to more apical regions of the cochlear duct. We found no evidence of any defect in inner hair cell (IHC) exocytosis or endocytosis using single hair cell recordings, nor any sign of hair cell synaptic abnormalities. Endocochlear potentials (EP) were normal. The mechanism underlying progressive hearing loss in these mutants remains elusive, but our findings of raised distortion product otoacoustic emission (DPOAE) thresholds and signs of OHC degeneration both suggest an OHC origin for the hearing loss. Synaptojanin2 is not required for normal development of hearing but it is important for its maintenance.

Bilateral third eyelid ectopic cilia in an adult horse

A 15-year-old Cob mare presented with a 4-month history of chronic epiphora and intermittent blepharospasm in the right eye. On ophthalmic examination, two translucent aberrant hairs were identified at the third eyelid margin corresponding to an area of corneal fibrosis and neovascularization. Partial excision of the third eyelid was performed, and histopathology confirmed ectopic hair follicles. Two weeks later, clinical signs recurred in the same eye. Examination revealed another pair of aberrant hairs on the bulbar surface of the third eyelid near its leading edge. This portion of the third eyelid was also excised, and histopathology confirmed two additional ectopic hair follicles. Eight months later, the horse developed similar clinical signs in the left eye. Ophthalmic examination showed a single aberrant translucent hair at the third eyelid margin associated with focal fibrosis and neovascularization of the ventromedial cornea. Partial excision of the third eyelid was performed, and histopathology confirmed an ectopic hair follicle within the third eyelid conjunctiva. Excision was curative at 4years postoperatively with no further recurrence in either eye.

Surface-enhanced Raman Scattering and X-ray Fluorescence Analyses of a Single Hair Colored with a Hair Dye Product.

This paper describes a method for obtaining information that can contribute to individual identification from a single hair colored with a hair dye product using a combination of surface-enhanced Raman scattering (SERS) and X-ray fluorescence (XRF) analyses. SERS and XRF spectra of single hog hairs colored with several commercially available hair dye products were measured. SERS spectral patterns tended to be different depending on the hair dye products used for hair coloring. However, SERS spectral patterns of single hog hairs colored with different hair dye products that would produce similar types of dyes were similar. By performing XRF analysis, characteristic metallic elements originating from some hair dye products were detected. Therefore, XRF can contribute to identifying the difference among colored hairs that cannot be identified only by SERS. SERS and XRF analyses of a single shed hair can contribute to individual identification used in forensic science.

Optimal processing for proteomic genotyping of single human hairs

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 μg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ± 5 to 73 ± 5 (p = 1 × 10−13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

A new method to estimate hair density of small mammal pelage

Abstract Hair density is the most important structural parameter contributing to insulation performance of mammalian pelage, and often is measured in ecophysiological, thermal biological, and evolutionary studies. To date, hair density has been measured using invasive methods on research objects; however, such methods remain challenging despite efforts to increase their ease of use. In this paper, we develop a new method to estimate hair density without skin sampling. We expressed hair density as the inverse of the number of hairs per unit area, that is, the surface area occupied by a single hair (Ah). This area could be further estimated by measuring distances between nearest neighboring hairs (Ln) and calculating the areas of triangles (A) defined by three randomly selected nearest neighboring hairs and representing half of Ah. Empirical tests using 11 skin samples from specimens of six small mammal species showed this to be a simple, lightly invasive, but accurate and widely applicable method.

<p><strong>Variability of leaf blade anatomical traits in the <em>Sesleria juncifolia</em> complex (Poaceae) on the Balkan Peninsula</strong></p>

This paper presents the results of a detailed study of leaf blade anatomical traits of populations of the Sesleria juncifolia complex from the Balkan Peninsula. The measurements were performed on cross sections of 302 tiller leaf blades from 24 populations. We calculated basic descriptive statistics for each character state. Principal component and canonical discriminant analyses were used to identify the structure of variability and the characters that majorly contributed to the differentiation of the defined groups. Cluster analysis was done to estimate the distances among the studied populations. We provide a detailed description of the leaf blade anatomy of the different populations of S. juncifolia complex investigated within Croatia, Bosnia and Herzegovina, Montenegro, Serbia and North Macedonia. The results show that most of the characters exhibit moderate degrees of variation. The principal component analysis shows slight separation of populations from northwestern Croatia. Canonical discriminant analysis shows that the three a priori defined groups—three species, S. interrupta, S. juncifolia and S. ujhelyii, can be distinguished based on leaf blade anatomical characters. In addition to the characters that were singled out as statistically most significant for differentiation of the three species in discriminant function analysis, the most useful characters for their delimitation are the length of the trichomes on the adaxial side of the leaf, as well as qualitative characters such as the sclerenchyma on the abaxial side of the leaf (continuous or interrupted) and the type of indumentum of the adaxial side of the leaf (very hairy, hairs long vs. very weakly hairy, hairs short). The northwestern populations of S. juncifolia mostly have glabrous leaves or single hairs on the adaxial side of the leaf, while in S. interrupta and S. ujhelyii populations, individuals always have hairy leaves. The presence of continuous subepidermal sclerenchyma has proven to be a good differential character for separating S. ujhelyii from the S. juncifolia and S. interrupta.

Proteomic Characterization of Damaged Single Hairs Recovered after an Explosion for Protein-Based Human Identification.

Evidence recovery is challenging where an explosion has occurred. Though hair evidence may be sufficiently robust to be recovered at the site, forensic analysis underutilizes the matrix by relying on morphological analysis. Where DNA is compromised, particularly in hair, protein-based human identification presents a promising alternative. Detection of amino acid polymorphisms in hair proteins as genetically variant peptides (GVPs) permits the inference of individualizing single nucleotide polymorphisms for identification. However, an explosive blast may damage hair proteins and compromise GVP identification. This work assesses effects of an explosive blast on the hair proteome and GVP identification, investigates microscopy as a predictor of proteome profiling success in recovered hairs to improve analysis throughput, and quantifies discriminative power in damaged hairs. The proteomics dataset has been deposited into the ProteomeXchange Consortium (PXD017427). With the exception of degradation in keratins K75, K80, K40, and keratin-associated protein KAP10-11 as markers of hair cuticular damage, corroborated by scanning electron microscopic analysis, minimal hair proteome degradation following explosion allowed successful proteome profiling of single hairs regardless of morphological damage. Finally, GVP identification remained independent of explosion conditions, permitting similar discriminative power between exploded and undamaged hairs. These findings lend greater confidence to GVP analysis in one-inch hairs for forensic identification and provide information about hair protein localization.

Liquid dispensing in the adhesive hairy pads of dock beetles.

Many insects can climb on smooth inverted substrates using adhesive hairy pads on their legs. The hair-surface contact is often mediated by minute volumes of liquid, which form capillary bridges in the contact zones and aid in adhesion. The liquid transport to the contact zones is poorly understood. We investigated the dynamics of liquid secretion in the dock beetle Gastrophysa viridula by quantifying the volume of the deposited liquid footprints during simulated walking experiments. The footprint volume increased with pad-surface contact time and was independent of the non-contact time. Furthermore, the footprint volume decreased to zero after reaching a threshold cumulative volume (approx. 30 fl) in successive steps. This suggests a limited reservoir with low liquid influx. We modelled our results as a fluidic resistive system and estimated the hydraulic resistance of a single attachment hair of the order of MPa · s/fl. The liquid secretion in beetle hairy pads is dominated by passive suction of the liquid during the contact phase. The high calculated resistance of the secretion pathway may originate from the nanosized channels in the hair cuticle. Such nanochannels presumably mediate the transport of cuticular lipids, which are chemically similar to the adhesive liquid.

Addendum to: Risk Stratification and Personal Protective Equipment Use in Pediatric Endoscopy During the COVID-19 Outbreak: A Single-center Protocol.

Addendum to: Risk Stratification and Personal Protective Equipment Use in Pediatric Endoscopy During the COVID-19 Outbreak: A Single-center Protocol.

Polarized Dermoscopy Facilitates Diagnosis and Treatment of Plantar Pseudohirsutism

Background. Plantar cutaneous conditions are the most common causes of foot discomfort limiting ambulation. Identification of indwelling foreign bodies is greatly facilitated by dermoscopy, allowing early detection of the character of the underlying pathological etiology. Furthermore, polarized dermoscopy provides ideal conditions of illumination and three- dimensional visualization of the involved site, without direct contact with the cutaneous surface. This unprecedented report describes its implementation for a solitary hair implanted into plantar skin. Case Presentation. A patient presented with localized plantar discomfort diagnosed as a soft tissue reaction to an embedded single human hair, folded and rooted inside the epidermis. Under magnified vision the phenomenon was characterized, the hair was extracted, the source was manipulated with a hypodermic needle and the lesion was removed with ensuing complete resolution. Conclusions. Previous reports of hair slivers embedded inside the skin were attributed to barbers or animal grooming and the treatment was dictated by the severity of the complications. In this relatively mild case the intervention was greatly facilitated by therapeutic dermoscopy. Therapeutic dermoscopy is advocated for use to evaluate and treat many diverse body surfaces, especially the plantar skin as it raises the likelihood of successful treatment of embedded foreign bodies. Precisely how a single hair may become lodged into normal plantar skin remains to be explained

High Spatial-Resolution Matrix-Assisted Laser Desorption/Ionization-Ion Trap-Time-of-Flight Tandem Mass Spectrometry Imaging for Depicting Longitudinal and Transverse Distribution of Drugs Incorporated into Hair.

This study aims to achieve high spatial-resolution tandem mass spectrometry (MS/MS) imaging for depicting longitudinal and transverse distribution of drugs in hair, which can provide indispensable information for the proper interpretation of hair test results, including the mechanism of drug incorporation into hair. Two types of hair samples were obtained and analyzed: User's Hair, sampled from a volunteer who took an over-the-counter medicine containing methoxyphenamine (MOP), a nonregulated analogue of methamphetamine; and Soaked Hair, prepared by soaking blank hair in MOP solution. Longitudinal and transverse-sectioning of single hair shafts was accomplished by freeze-sectioning using customized microtomes. Vapor deposition of α-cyano-4-hydroxycinnamic acid provided the finest matrix layer (resolution <1 μm, 0.7-μm thickness), although it provided less effective ionization of MOP compared to aerosol spraying or a combination of both. Matrix-assisted laser desorption/ionization (MALDI)-ion trap (IT)-time-of-flight (TOF) MS/MS permitted the imaging of trace-level MOP in hair with a MS/MS window setting of ±0.02 Da and a spatial resolution setting at 5 or 10 μm. For Soaked Hair, localization of MOP in the peripheral part was clearly depicted, but no such biased distribution was observed in the transverse sections of User's Hair. MOP-positive bands generated corresponding to the time periods of MOP intake could be observed on the longitudinal sections of User's Hair. This method can provide forensically crucial information regarding hair analysis for drugs: drug incorporation mechanism into hair, discrimination of undesired surface contamination from endogenous incorporation of ingested drugs, and precise elucidation of drug-use history.

Proteomic genotyping: Using mass spectrometry to infer SNP genotypes in pigmented and non-pigmented hair

Proteomic genotyping uses genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of corresponding non-synonymous SNP alleles. We have focused on hair proteins as a source of protein-based genetic information in a forensic context. An optimized sample processing protocol for hair shafts has been developed for use on a single hair that allows us to conduct validation protocols on real world samples. This includes whether the inferred SNP genotypes are robust and not systematically affected by biological or chemical variation in hair proteomes that might be obtained from a crime scene. To this end we analyzed the hair of 4 mature individuals with a mixture of pigmented and non-pigmented hair. We demonstrate significant changes in the proteomes of grey versus pigmented hair. Vesicle specific proteins and lipid catabolism proteins were enriched in pigmented hair, and housekeeping proteins and lipid anabolic enzymes were enriched in grey, non-pigmented hair. The resulting profiles of genetically variant peptides, however, were more correlated with profiles from the same individuals regardless of pigmentation status. Together with other published evidence, this finding indicates that profiles of genetically variant peptides are robust and more correlated with other genetically variant peptide profiles from the same individual irrespective of changes occurring in the hair protein profile. Based on this small sample, investigators using profiles of genetically variant peptides to infer random match probabilities should not expect to observe differences based on the pigmentation of the hair shaft.

Kallmann syndrome in a 17-year-old boy

Aim. To describe a rare hereditary disease — Kallmann syndrome — in a 17-year-old boy.Materials and methods. A retrospective analysis of anamnestic information, the course of the disease, laboratory and instrumental data and treatment of a 17-year-old patient with Kallmann syndrome was carried out. The patient underwent treatment in a gastroenterological department of the Children’s Regional Clinical Hospital in Krasnodar in March 2019.Results. Patient K., 17 years old, was admitted to the gastroenterological department of the Children’s Regional Clinical Hospital in March 2019 with complaints of weakness, nausea, “hungry” abdominal pain and decreased appetite. Upon examination, the child revealed duodenal ulcer. However, an in-depth examination found that the boy was lagging behind in physical and sexual development. In this connection, an endocrinological examination was performed, which discovered: a decrease in the concentration of sex hormones relative to the age norm (luteinizing hormone, follicle-stimulating hormone, testosterone), delayed bone age as compared to the passport age, discrepancy between the testicular size and the patient’s age, olfactory bulb hypoplasia according to brain MRI, 1st degree smell disorder, the presence of a mutation in the KAL1 gene. The boy was diagnosed with: Kallmann syndrome, X-linked recessive inheritance. Complications: hypogonadotropic hypogonadism. The chosen treatment included prolonged testosterone esters for parenteral administration of 250 mg once per 3–4 weeks. Following 6 months of therapy, positive signs were observed: an increase in height by 2 cm, an increase in testicular volume by 3 ml, the appearance of single hairs at the base of the penis.Conclusion. Kallmann syndrome is a rare pathology, whose main manifestations include delayed sexual development and hypo- or anosmia. For a timely diagnosis and treatment, it is of great importance to identify symptoms and conduct a comprehensive examination. Timely initiation of hormonal treatment allows puberty to occur in all cases, thus facilitating social and psychological adaptation of such patients.

Warty dyskeratoma; A rare entity with involvement of multiple adjoining follicles

Warty dyskeratoma is a rare epidermal tumor which occurs in middle aged or older persons, clinically manifests as papule or nodule and involves head and neck region in most of the cases. 65y F presented to the OPD with complaint of nodular swelling over the right cheek with itching for 15 years. On clinical examination patient had 3x1.8 cm nodule, firm in consistency, mobile, with ulceration over tumor. Routine investigations were within normal limits and Fine Needle Aspiration Cytology was reported as suspicious of malignancy. Histopathology of excised mass revealed hyperkeratotic, parakeratotic and acanthotic epidermis show large multiple invaginations comprising of lacunae along with villi formation. Stratum corneum and upper malpighi layer shows abundant dyskeratotic acantholytic cells. As per literature these changes are usually noticed in single hair follicle. Here we describe a rare case of warty dyskeratoma with multiple adjacent hair follicles involvement.

Simultaneous analysis of typical halogenated endocrine disrupting chemicals and metal(loid)s in human hair

Hair analysis has been an important approach in evaluating population exposure to various environmental factors. To meet the requirements of human environmental epidemiology studies, we aimed to develop an efficient method for simultaneous analysis of various metal(loid)s and some typical environmental halogenated endocrine disrupting chemicals (hEDCs) (i.e., polychlorinated biphenyls, polybrominated diphenyl ethers, and organochlorine pesticides, as well as some of their hydroxyl substituted metabolites) in a single hair sample. The hair was washed successively with surfactant solutions, methanol solvent, and deionized water to remove impurities attached to the hair surface. Efficiency was comprehensively compared among various washing strategies. The hair sample was further pulverized into fine powder with a median diameter (25th–75th percentile) of 8.6 (5.9–13.5) μm. The hair organic components were extracted by acetonitrile solvent and compared with the microwave-assisted extraction method. The hEDCs in the supernatant acetonitrile phase were quantified by gas chromatography–mass spectrometry, and the metal(loid)s in the precipitate hair were further analyzed by inductively coupled plasma mass spectrometry. Our developed method was further applied to analyze the hair samples of 165 pregnant women. The results showed that particles attached to the surface of the hair could not be washed off completely. However, we proposed a protocol framework to wash hair with relatively high efficience, which includes warm water incubation, and use of surfactant and organic solvent. The recoveries of the concerned hEDCs and metal(loid)s were overall in the range of 80% to 120%. For the women population, the method can efficiently recognize the typical exposure characteristics of the concerned hEDCs and metal(loid)s. Our study significantly ameliorated the deficiencies of the traditional hair washing strategy and developed an efficient method for simultaneous analysis of various metal(loid)s and hEDCs in a single hair sample. This method will provide important support for population complex exposure analysis and facilitate environmental exposome studies.

Using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for investigations on single hair samples to solve the contamination versus incorporation issue of hair analysis in the case of cocaine and methadone

Drug testing in hair is a controversial subject of discussion. Claims that decontamination protocols could generate false-positive samples, by washing contamination in hair, have unsettled many toxicologists. At least for zolpidem (known for showing only minor contamination), it could be shown that differentiation of the drug incorporated via the bloodstream from contamination was possible. The current work addresses cocaine and methadone, known for their high concentrations and contamination issues. Longitudinally and cross-sectioned samples of drug-soaked hair, consumer hair and cocaine powder contaminated hair were investigated using time of flight-secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). In addition, the resulting wash solutions were investigated using LC-MS/MS. Differentiation of contamination from incorporation was possible for soaked and consumer hair samples. Therefore, contamination could be localized in the superficial compartments of hair and could be removed using strong wash protocols. In the case of powder contaminated hair samples, a small amount of cocaine remained in the inner structures even after the application of the strongest wash protocols. However, taking into consideration the differences in their behavior during decontamination steps compared to both soaked and authentic hair samples, the validity of this contamination protocol (rubbing cocaine powder into hair) must be questioned. Furthermore, when using cut-off values and metabolite ratios (from routine hair analysis), the differentiation of incorporation from contamination was possible also for all our experimental samples in this study. Inclusion of metabolites and application of cut-off values are therefore a must in routine hair analysis.