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| ORIGINAL ARTICLE |
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| Year : 2019 | Volume
: 5
| Issue : 1 | Page : 20-23 |
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A comparative study of human and animal hairs: Microscopic hair comparison and cytochrome c oxidase I species identification
Venusia Cortellini, Anna Carobbio, Gloria Brescia, Nicoletta Cerri, Andrea Verzeletti
Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Forensic Medicine Unit, University of Brescia, Brescia, Italy
| Date of Web Publication | 28-Mar-2019 |
Correspondence Address:
Dr. Venusia Cortellini
Piazzale Spedali Civili 1, 25123 Brescia
Italy
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/jfsm.jfsm_49_18
Human and animal hairs have been used in forensic investigations for over a century. Hair is stable under adverse natural conditions; hence, it is often recovered at the crime scene, and it is necessary to determine whether the hair is of human or animal origin. Morphological and genetic characteristics are useful to differentiate human hair from animal hair. In the present study, we analyzed the distinguishing characteristics of hair of various species. In addition, we explore species identification by cytochrome c oxidase I mitochondrial gene analysis. We confirm that both the microscopic and molecular analyses of hairs are useful in forensic investigations.
Keywords: Cytochrome c oxidase I, hair, optical microscope, species identification
How to cite this article:
Cortellini V, Carobbio A, Brescia G, Cerri N, Verzeletti A. A comparative study of human and animal hairs: Microscopic hair comparison and cytochrome c oxidase I species identification. J Forensic Sci Med 2019;5:20-3 |
How to cite this URL:
Cortellini V, Carobbio A, Brescia G, Cerri N, Verzeletti A. A comparative study of human and animal hairs: Microscopic hair comparison and cytochrome c oxidase I species identification. J Forensic Sci Med [serial online] 2019 [cited 2020 Oct 31];5:20-3. Available from: https://www.jfsmonline.com/text.asp?2019/5/1/20/255132 |
| Introduction | |  |
During the course of a criminal investigation, many types of physical evidences are encountered. One of the most common is hair evidence. A hair can be defined as a slender, thread-like protein outgrowth from a follicle in the skin of mammals. Hair grows from the papilla, which is a metabolically active structure. The shaft of hairs is made up of dead, cornified cells that project outward from the skin and a root that is imbedded in the skin. Composed mainly of keratin, it has three morphological regions: the cuticle, the medulla, and the cortex. The cuticle is a translucent outer layer of the hair shaft consisting of scales covering the shaft. The medulla is a central core of cells that may be present in the hair. If it is filled with air, it appears as a black or opaque structure under transmitted light or as a white structure under reflected light. The cortex is the main body of the hair composed of elongated and fusiform cells. It may contain cortical fusi, pigment granules, and/or large oval-to-round-shaped structures called ovoid bodies.
The identification and comparison of human and animal fur can provide investigators with valuable information for potential leads. Microscopic comparison is considered a reliable tool for the evaluation and specific identification of the characteristics found in hair. Mitochondrial DNA (mtDNA) testing can also provide additional information. The combination of these technologies profoundly affects the way forensic scientists, investigators, and prosecutors view hair evidence.[1] Because DNA technologies are time- and cost-consuming, the first step in the analytical process is the microscopic examination[2],[3] and determination whether the hair is of human or animal origin.[4] Microscopy permits a large number of questioned hairs collected from evidence to be examined quickly, thus minimizing the time and cost of the mtDNA analyses. The microscopic comparison of morphological characteristics of hair has been accepted both scientifically and legally for decades. There are many differences in human and animal hairs such as color, tip, root, cuticle, scale, medulla, cortex, and other special characteristics.[5] In the present study, we analyzed distinguishing characteristics that can be used to differentiate human hair from animal hair and to discern between animal species.
Sequence analysis of mtDNA was also performed for genetic species identification. The identification of animal biological diversity was proposed on a large scale through the use of one-short, conserved mtDNA region cytochrome c oxidase I (COI) mitochondrial gene, the “DNA barcode.”[6] This DNA barcoding has become a method for species identification across a broad range of eukaryotic taxa by sequencing the standardized short mtDNA fragment. In this study, we also applied this current method for species identification from hair employed for microscopic examinations, to confirm the preliminary taxonomic distinction obtained by microscopic characterization.
| Materials and Methods | | |
Samples collected
The study was conducted on the following seven different species from Northern Italy: Homo sapiens (human), Bos taurus (cow), Canis lupus familiaris (dog), Capra hircus (goat), Equus ferus caballus (horse), Felis catus (cat), and Oryctolagus cuniculus (rabbit). We plucked a tuft of hair from the scalp of ten representative individuals of each species studied.
The animal hairs discussed are limited to those animals most likely to be examined in our laboratory in forensic casework.
Sample preparation
All the samples were put in serially marked paper envelopes immediately after they were collected and then stored at room temperature. The same day, each hair sample was cleaned with distilled water to remove dust and debris, if any, and left to dry until the next day.
Mounting of hair strands and microscopic examination
Examination of the overall hair morphology
One strand of hair with root from each individual was placed on a microscope slide and cover slipped, for a total of seventy samples. The hair was scanned along the length at 10X, 25X, and 40X under an optical microscope to observe the morphological characteristic of the tip and root.
Examination of hair cuticle scale patterns
It is very difficult to directly observe scale pattern from hair strand on a slide. Hence, a cast was made using nail polish to obtain the impression of the scales. A thin layer of nail polish was spread on a microscope slide, and a hair was placed in the middle of the slide. The polish was allowed to harden, and the hair was gently removed.[7] Then, the scale pattern was observed under an optical microscope at 10X, 25X, and 40X.
Examination of hair cross sections
To characterize the medulla and the cortex, one single hair of all different species was formalin fixed and paraffin embedded. After fixation, the samples were cut in 4-μm-thick sections on a microtome with a disposable blade. The longitudinal and transverse sections were observed under an optical microscope at 10X, 25X, and 40X.
Cytochrome c oxidase I analysis
For DNA analysis, the fixed hairs were deparaffinized in two changes of xylene for 10 min; rehydrated in 100% ethanol, 90% ethanol, and 70% ethanol for 5 min each; and finally rinsed in RNase-free H2O. DNA was extracted from each hair sample using QIAamp® DNA Micro Kit (QIAGEN, Hilden, Germany), following the manufacturer's protocols for forensic casework samples. The COI region of mtDNA was amplified according to Ferri et al.,[8] using two sets of specific primers (F220 and R220, F439 and R439). Purified samples were directly placed on Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems, California, USA). Sequences were resolved in Sequencing Analysis v 6.0 software (Applied Biosystems), and the obtained sequences were imported into Barcode of Life Database (BOLD)[9],[10] search engine to determine the species.[11],[12],[13]
| Results | | |
The purpose of this study was to catalog the distinguishing characteristics of different species by optical microscope to differentiate human hairs from animal hairs and to discern between animal species. Many differences in human and animal hairs were found. The results of the microscopic examination of the hair samples are shown in [Table 1]. We were able to speciate all hair samples by microscopic features alone.
We have assembled a photomicrograph collection that can be used to differentiate human hair from animal hair and to discern between animal species using the roots [Figure 1], cuticles [Figure 2], and medulla and cortex [Figure 3] and [Figure 4] for all the species analyzed. | Figure 1: Photomicrograph of Homo sapiens (a), Bos taurus (b), Canis lupus familiaris (c), Capra hircus (d), Equus ferus caballus (e), Felis catus (f), Oryctolagus cuniculus (g) hair root
Click here to view |
 | Figure 2: Photomicrograph of Homo sapiens (a), Bos taurus (b), Canis lupus familiaris (c), Capra hircus (d), Equus ferus caballus (e), Felis catus (f), Oryctolagus cuniculus (g) hair cuticle
Click here to view |
 | Figure 3: Photomicrograph of Homo sapiens (a), Bos taurus (b), Canis lupus familiaris (c), Capra hircus (d), Equus ferus caballus (e), Felis catus (f), Oryctolagus cuniculus (g) hair medulla and cortex (transverse section)
Click here to view |
 | Figure 4: Photomicrograph of Homo sapiens (a), Bos taurus (b), Canis lupus familiaris (c), Capra hircus (d), Equus ferus caballus (e), Felis catus (f), Oryctolagus cuniculus (g) hair medulla and cortex (longitudinal section)
Click here to view |
To confirm the preliminary taxonomic distinction obtained by microscopic characterization, the COI mtDNA regions were successfully amplified. The sequences obtained from each hair were then aligned in the BOLD database; the results reported confirmed the proper speciation as we had determined (data not shown).
All of the morphologic characteristics observed for the individuals of the same species were identical, except color.
Due to the limited number of individuals studied, the observed morphologic results may not be generalized.
| Conclusions | | |
Both the microscopic and molecular analyses of hairs are useful in forensic investigations. Microscopic analysis of hairs demonstrates morphological differences that enable discrimination between human hair and animal hair and among animal species. Genetic analysis confirmed the diagnosis of species prior to the microscopic analysis. Furthermore, we demonstrated that treatment carried out for microscopic observation, formalin fixed and paraffin embedded and deparaffinized in xylene, does not compromise the subsequent genetic investigation. The use of optical microscope to observe hair from humans and other species, therefore, allows a first screening for the hair selection to be submitted to DNA analysis, allowing a saving of time and costs, thus avoiding the genetic molecular analysis of nonhuman hair. We can also confirm the ability to speciate hairs by microscopy alone, when the genetic profile is incomplete or inconclusive. However, we suggest both techniques must be used to confirm the species of the sample.
Acknowledgment
The authors thank Dr. Heitor Correa for his help in English revision.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Houck MM, Budowle B. Correlation of microscopic and mitochondrial DNA hair comparisons. J Forensic Sci 2002;47:964-7.  |
| 2. | Bisbing RE. The forensic identification and association of human hair. In: Saferstein R, editor. Forensic Science Handbook. Vol. I. Englewood Cliffs, NJ: Prentice Hall; 2002. |
| 3. | Bisbing RE. Human air in a forensic perspective. In: Proceedings of International Symposium on Forensic Hair Comparisons. Quantico, VA: FBI Academy; 1985. |
| 4. | Kshirsagar SV, Singh B, Fulari SP. Comparative study of human and animal hair in relation with diameter and medullary index”. Indian J Forensic Community Med 2009;2:105-8. |
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| 6. | Wilson-Wilde L, Norman J, Robertson J, Sarre S, Georges A. Current issues in species identification for forensic science and the validity of using the cytochrome oxidase I (COI) gene. Forensic Sci Med Pathol 2010;6:233-41. |
| 7. | Robertson J. Forensic and microscopic examination of human hair. In: Forensic Examination of Hair. London: Taylor and Francis; 1999. |
| 8. | Ferri G, Alù M, Corradini B, Licata M, Beduschi G. Species identification through DNA “barcodes”. Genet Test Mol Biomarkers 2009;13:421-6. |
| 9. | Ratnasingham S, Hebert PD. A DNA-based registry for all animal species: The barcode index number (BIN) system. PLoS One 2013;8:e66213. |
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| 11. | Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identification through DNA barcodes. Proc R Soc Lond B 2003;270:313-21. |
| 12. | Hajibabaei M, Singer GA, Hebert PD, Hickey DA. DNA barcoding: How it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet 2007;23:167-72. |
| 13. | Hebert PD, Ratnasingham S, deWaard JR. Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci 2003;270 Suppl 1:S96-9. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1]
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