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Forensic Science International 178 (2008) e49e52

Letter to the Editor

Response to comments on Morphological changes in human head hair subjected to various drug testing decontamination strategies by Hill et al. We would like to thank Ms. Hill and her colleagues at Psychemedics Corporation for their valuable comments and the opportunity to clarify both the purpose of our study and selected issues. Before addressing the issues, we would like to clarify that the hair locks used in our studies reported in the Journal of Analytical Toxicology [1] and in Forensic Science International [2] were from the same sources. Hill et al. remarked that we did not include micrographs of all ve hair types in the original publication. We did this in the interest of conserving space in the article. We have included representative micrographs at 1000 of all ve hair samples prior to any treatment with this reply. As previously stated and as can be seen here, the ve hair samples prior to decontamination treatments were very similar in appearance. In our evaluation of the effects of the decontamination protocols on hair, we chose at random 10 hair strands from each of the hair types for each of the protocols. The individual hairs were mounted on carbon adhesive on the stage stubs to allow observation of the mid-shaft regions. For hair types 1, 2 and 4, we observed that one hair strand out of the 10 was broken as depicted in Figure 7. In our sampling, we observed breaks like this only in strands decontaminated using aqueous buffer. Hair locks from these groups decontaminated with aqueous buffers were noticeably more brittle when handled. All hair strands observed from the aqueous buffer treatment exhibited the appearance of degrading scales as shown in Figures 5 and 6. Three of the 10 aqueous treated bers observed had the stripped appearance of the ber pictured in Figure 8. For the methanol and methylene chloride treatments, all hair strands observed exhibited some degree of scale lifting as depicted in Figures 3 and 4.

DOI of original article: 10.1016/j.forsciint.2008.03.004. 0379-0738/$ see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2008.03.005


Letter to the Editor / Forensic Science International 178 (2008) e49e52

Hill et al. identied the electron-dense bodies in the cuticle shown in Figure 10 as dye inclusions. We are unaware of any references describing the scanning electron microscopy (SEM) appearance of dye inclusions in the cuticle and therefore cannot rule out this possibility. However, the reference cited by Hill et al. does not address this issue. Instead, it discusses visualizing dyed hair by optical observation of the hair cross section and we quote: For forensic or commercial evaluations, light microscopy can readily determine whether or not hair has been dyed with a permanent dye or has been recently dyed with a semi permanent dye. Human scalp hair does not generally contain pigment in the cuticle. Therefore, a light microscopic evaluation of ber cross sections or even an optical section can readily reveal whether the hair in question has been treated with a commercial hair dye determining whether the cuticle has been dyed. ([3, p. 313]) The light micrograph related to the passage in Robbins [3], Figure 6-2, depicts cross sections of dyed hairs demonstrating a uniform circumferential staining of the cuticle and not a pattern consistent with our observations of a localized collection of ovoid electron-dense bodies as shown in Figure 10 of our article. Melanosomes are known to be electron-dense (dark appearance in electron microscopy) and of this ovoid shape. Hill et al. are correct that Robbins [3] reports melanosomes to be 0.20.8 mm. However, Nanko et al. [4] provide electron micrographs with ovoid, electron-dense bodies ranging from 0.2 mm to larger than 1 mm, which they identify as melanosomes. This is consistent with our observations. While melanosomes in the cuticle are not common, Robbins [3] indicates that they are a possibility.

Letter to the Editor / Forensic Science International 178 (2008) e49e52


Hill et al. discuss the use of methylene blue as an alternative method of assessing the condition of the hair. As was discussed in the article, we used hair that had not been subjected to bleaching, coloring, permanent waves, or other cosmetic treatments, according to the hair donors and the cosmetologist who provided the hair samples. We also evaluated each hair sample used in this study by visualizing the hair structure using SEM. Hill et al. advocate the use of methylene blue staining of the hair to determine functional porosity. The reference that Hill et al. have previously cited for use of this method [5] provides a cursory description of methylene blue staining of the hair, but provides no criteria by which to objectively evaluate the hair. Their more current reference [6] does provide more information about methylene blue staining, but it is still a strictly qualitative method. Their references do not include a validation of the efcacy of methylene blue for determining the functional porosity, nor a denition of functional porosity based upon methylene blue staining. We are unaware of any publications validating the efcacy of methylene blue staining as a measure of functional porosity, but are aware of its use in the cosmetic industry to evaluate hair dyes [3]. The images presented by Hill et al. [6] indicate highly variable staining even within individual. Thus, methylene blue and SEM likely are equivalent in their limited ability to determine damage or porosity of the hair. Weathering and chemical damage can result in damage to the cuticle as observed in several reports we cited [79]. We are not suggesting that the damage to the hair from chemical treatment or extended weathering is directly comparable to an aqueous wash, but noting that we observed similar morphological changes after aqueous washing. Nanko et al. [4] reported, based on SEM and transmission electron microscopy (TEM) observations of hair from Japanese swimmers, that some hairs exposed to the water exhibited complete loss of the cuticle. The accompanying micrographs are quite similar in appearance to our Figures 8 and 9. The cross sectional TEM gures clearly show the loss of the cuticle, which the authors attribute to friction with the water. Thus, there is signicant evidence that the hair cuticle can be disrupted by exposure to water, especially when the water moves through or over the hair, as would be the case in the aqueous decontamination treatment. The following is provided in reference to the questions we posed in our article and the responses provided by Hill et al.: (1) Do decontamination procedures remove metabolically deposited drug? and (3) Are decontamination protocols effective in removing external contaminants on hair when normal hygienic practices are followed or when hair has been cosmetically treated? These questions cannot be answered by observing analytes that are not true metabolic markers, providing denitive evidence of drug ingestion. A true metabolic marker is a compound whose presence in the body can only be explained by enzymatic biotransformation of the drug. We have reported the presence of benzoylecgonine and norcocaine in 25 illicit cocaine samples complicating the interpretation of previous studies especially for cocaine [10]. Janzen [11,12] reported upward of 20% cocaethylene in

cocaine smuggled in ethanolic drinks and Casale [13] provides a discussion of the extent and pathways of cocaethylene presence in illicit cocaine. The interpretation is also complicated by the potential for a true metabolic marker to be deposited on the hair from sweat or sebum and not removed by decontamination treatments. Therefore, although Hill et al. reported that they were able to correctly identify the contaminated hair samples in our previous study using their companys cocaine analyte ratio and wash criteria, concern remains that the analytes used are not true metabolic markers and, therefore, may have entered the hair by routes other than enzymatic biotransformation and confound the interpretation of the reported results. (2) Is contaminated drug redistributed during washing? Thermodynamically, any matrix may experience redistribution of small molecules that are not covalently linked to the matrix. In hair, this might occur during decontamination, cosmetic treatments (shampooing, applying hair thickeners or styling products), or exposure to other solvents. This is still unanswered. (4) Do hair color and the presence of melanosomes in the hair cuticle have an impact on the effectiveness of decontamination procedures? The structures we showed in Figure 10 may or may not be melanosomes. As we noted, our observations were consistent with those by Nanko et al. [4], who identied ovoid electron-dense bodies ranging from 0.2 mm to larger than 1 mm as melanosomes. Without better information of the prevalence of melanosomes in the cuticle, this question is unresolved. To our knowledge, our publication is the rst to attempt to describe the morphological changes occurring in the hair due to decontamination strategies commonly employed in hair testing. The issues of variable porosity and how to characterize it are important considerations in reaching a valid conclusion about the route of drug exposure for any given individual. As we have discussed previously, the best denitive measure to address the potential for contamination is the identication of true metabolic markers. Such markers would greatly reduce the potential argument that drug is present in hair due to environmental contamination. Until such point, there will remain discussion about the validity of various models for contamination and the continued demonstration that drug present in the environment can associate with hair and potentially be confused with ingested drug. We encourage all researchers and all members of the hair testing industry to continue research efforts to identify metabolic markers for drugs of interest. This advance would certainly improve forensic interpretation of results from future hair tests for drugs of abuse. References
[1] P.R. Stout, et al., External contamination of hair with cocaine: evaluation of external cocaine contamination and development of performancetesting materials, J. Anal. Toxicol. 30 (8) (2006) 490500. [2] P.R. Stout, et al., Morphological changes in human head hair subjected to various drug testing decontamination strategies, Forensic Sci. Int. 172 (2007) 164170.


Letter to the Editor / Forensic Science International 178 (2008) e49e52 [12] K. Janzen, Ethylbenzoylecgonine: a novel component in illicit cocaine, J. Forensic Sci. 36 (4) (1991) 12241228. [13] J.F. Casale, Cocaethylene as a component in illicit cocaine, J. Anal. Toxicol. 31 (3) (2007) 170171.

[3] C.R. Robbins, Chemical and Physical Behavior of Human Hair, 4th ed., Springer, New York, 2002. [4] H. Nanko, et al., Hair-discoloration of Japanese elite swimmers, J. Dermatol. 27 (10) (2000) 625634. [5] W.A. Baumgartner, V.A. Hill, Sample preparation techniques, Forensic Sci. Int. 63 (13) (1993) 121135, discussion 13743. [6] V. Hill, T. Cairns, M. Schaffer, Hair analysis for cocaine: factors in laboratory contamination studies and their relevance to prociency sample preparation and hair testing practices, Forensic Sci. Int. (2007). [7] B.S. Chang, et al., Ultramicroscopic observations on morphological changes in hair during 25 years of weathering, Forensic Sci. Int. 151 (23) (2005) 193200. [8] C. Scanavez, I. Joekes, H. Zahn, Extractable substances from human hair: a discussion about the origin of the holes, Colloids Surf B Biointerf. 39 (1/ 2) (2004) 3943. [9] H.J. Ahn, W.S. Lee, An ultrastuctural study of hair ber damage and restoration following treatment with permanent hair dye, Int. J. Dermatol. 41 (2) (2002) 8892. [10] J.D. Ropero, et al., K2 Signature analysis of 25 Illicit Cocaine Samples and a Comparison to Analysis by AccuTOF-DART, American Academy Forensic Sciences, Washington, DC, 2008. [11] K. Janzen, Concerning norcocaine, ethylbenzoylecgonine, and the identication of cocaine use in human hair, J. Anal. Toxicol. 16 (6) (1992) 402.

Peter R. Stout* Jeri D. Ropero-Miller Michael R. Baylor John M. Mitchell RTI International, Center for Forensic Sciences, 3040 Cornwallis Road, P.O. Box 12194, Research Triangle Park, NC 27709, United States *Corresponding author. Tel.: +1 919 316 3450; fax: +1 919 541 7042 E-mail address: Available online 2 May 2008