Microscopy of Hair Part 1: A Practical Guide and Manual for Human Hairs

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Methods of Hair Recovery

Sir Edmund Locard's principle (1930) states that "whenever two objects come into contact, a transfer of material will occur. Trace evidence that is transferred can be used to associate objects, individuals, or locations" (Scientific Working Group on Materials Analysis 1999). Because of the nature of trace evidence, when processing evidentiary items, care should be taken to minimize the possibility of contamination and cross-transfer. Examinations should be sequenced to maximize the potential value of the submitted evidence.

Hairs can be recovered from evidentiary items using a number of different techniques. Some of the methods used to collect hairs from clothing and bedding items are scraping, shaking, taping, and picking. Debris from large carpeted surfaces might be vacuumed into a filtered canister. If the specific location of a hair on a clothing item is important, it might be necessary to pick off the hair or tape the item and record where the hair was removed.

Whichever method is used, it should be done in a location designed for that purpose to avoid the possibility of contamination and cross-transfer. Special lighting and magnification may facilitate the location and recovery.

Scale Casts

It may be necessary to make a scale cast of the hair specimen in order to see the scale pattern more clearly, particularly in the identification of some animal hairs. Ogle and Mitosinka (1973) devised a quick and easy method of making a scale cast with the use of a Polaroid film-print coater. A thin layer is applied to a glass microscope slide with two or three passes of the Polaroid print coater. The hair specimen is lightly pressed onto the film and allowed to stand until the film is dry. The hair is then pulled from the film, and the cast remains.

A method developed by Crocker (1998) at the Centre of Forensic Sciences in Toronto, Canada, uses clear tape as a mounting medium and coverslip together, which allows for quick observation of such surface features as the scale pattern.

Scale casts may also be prepared using clear nail polish. A thin coat is painted on a glass microscope slide or, if the lacquer is thinned with acetone, a drop may be allowed to run down the surface of the slide. The hair is placed on the slide and allowed to dry. When the surface has dried, the hair is removed to reveal the scale pattern.

Sampling Methods

After trace debris has been removed from items of evidence, it is necessary to select the appropriate types and number of hairs for examination. Sometimes when removing a large quantity of debris (e.g., vacuuming), it may be necessary to select only a representative sample. This process includes selecting samples such as hairs of different lengths, racial groups, body area, and color. Another method is to select hairs that are similar in appearance to a target group (e.g., known hairs from a suspect or victim). The combination of random and target sampling ensures a representative sample.

Selecting hairs for microscopic analyses takes place during the initial processing as well as during low-power microscopy at the bench. The microscopic characteristics of hairs are viewed and selected with the intention of providing an examiner with a good range of the hair types present.

Head hairs and pubic hairs exhibit a greater range of microscopic characteristics than other human hairs; therefore, head and pubic hairs are routinely forensically compared. An adequate selection of known hair samples includes both random pullings and combings. The number of hairs necessary to represent a suitable known sample varies with the individual. Twenty-five randomly selected head hairs are generally considered adequate to represent the range of hair characteristics of that individual. It is recommended that the same number of hairs be collected from the pubic region. The selection of hairs to be mounted from a known hair standard may be random, but representative, especially when the known standard consists of many hairs.

The collection of known head hair standards from a suspect might take place many months, possibly years, after the crime. In these instances, the characteristics of the known head hair sample may look quite different from hairs that were shed when the crime occurred. Some hair examiners have indicated that a one-year time span is the outside limit, and environmental conditions or cosmetic alterations could make it shorter. Pubic hairs seem to retain their characteristics for a longer period of time.

Glass Microscope Slide Preparation

Hair specimens are prepared for microscopic examination by mounting them in a semipermanent medium, such as Permount®. Whatever mounting medium is selected, the refractive index of the medium should approximate that of hair (1.52) in order to visualize the internal microscopic characteristics.

Positioning a hair on the glass slide is made easier by first applying a thin film of solvent on the slide surface. Longer hairs are configured in a figure eight in order to fit it under the cover slip. This enables the examiner to view the entire hair from root to tip. One or more hairs can be mounted on a slide, depending on their thickness and curl. Too many hairs on one slide can cause excessive overlapping that may obscure the viewing of characteristics of underlying hairs. Excess solvent can be removed with a small square of blotter paper. Several drops of mounting medium are applied on top of the hair(s), and a cover slip is carefully lowered to prevent the presence of air bubbles. Figure 23 diagrams this process. It may be necessary to apply some weight to the cover slip in order to ensure a thin mount. The thinner the mount, the easier it is to examine the hairs.

Figure 23.

Illustration of

Slide Preparation

In most cases, hairs can be mounted directly onto the slide; however, occasionally, in order to observe structural detail, it may be necessary to clean debris from the hairs. If covered with blood, hairs can be cleaned with a saline solution, but because water is not miscible in Permount®, the sample must be dried completely before applying the mounting medium. Oily or other debris-contaminated hairs can be cleansed with xylene or an ether-alcohol solution. Before cleaning the hairs, consider if any blood and other materials on the surface may have evidentiary value.

Microscope

Conducting a reliable hair examination involves maintaining a reliable microscope. It must be kept in good working condition and adjusted for proper illumination. General care and cleaning procedures should be followed to prevent dust, dirt, fingerprints, or other contaminants from affecting the use of the microscope.

Illumination

The specimen field is illuminated with a low-voltage tungsten filament lamp. A color-correcting blue filter is used to approximate white light. If each side of the comparison microscope uses a separate light source, it is essential to color-balance the light sources before starting any comparison. Calibrating for uniform illumination between the sides of the comparison microscope and in each field of view should be done daily.

Köhler illumination ensures that the light path is optimized. A modified Köhler illumination calibration process is as follows:

1. Open the field and aperture diaphragms

2. Adjust the interpupillary distance of the oculars

3. Place the specimen on the stage and focus using the nonadjustable eyepiece, then use the adjustable eyepiece to focus that eyepiece

4. Close the field diaphragm by half

5. Focus and center the condenser

6. Open the field diaphragm until it is just out of view

7. Remove the eyepiece and close the aperture diaphragm by 1/3

8. Replace the eyepiece

 

The microscope is ready to use.

Field Diaphragm

The field diaphragm protects the specimen against unnecessary heating. As part of Köhler illumination, the field diaphragm is closed, and then opened until it just clears the field of view. If it is opened too far, the excess light will cause the image to lose its sharpness and contrast.

Aperture Diaphragm (condenser)

The aperture diaphragm determines the resolution and contrast of the microscopic image. To observe specimens under normal contrast, remove the eyepiece and reduce the objective aperture by one-third.

Substage Condenser

The condenser lens concentrates the light on the specimen. It should be in position with the objective at a numerical aperture (N.A.) larger than 0.25. The condenser lens may be swung out of position with the objective having an N.A. of less than 0.25.

Objective Lens

The objective lens forms an inverted and side-reversed intermediate image. On each objective are sets of numbers. The first set refers to the mechanical tube length, which includes the thickness of the cover glass for which the objective was designed (in mm). The next set indicates the magnification of the objective lens and the numerical aperture. Letters before the magnification identify the type of system. Apo refers to apochromatic, Fl stands for fluorite, or Oel refers to oil immersion. If nothing is listed before the magnification, the system is achromatic.

Numerical Aperture

The numerical aperture is an important aspect of the microscope; it determines the resolving power or the ability of the objective to focus on the separate features in a sample. It is the relationship between the widest angle of the path light travels to the objective, and the refractive index of the mounting medium that the light passes through. This relationship determines the maximum resolution of the objective. The ability of the objective lens to make fine structural detail in the specimen distinct is the main purpose for using a microscope, thus its dependence on the numerical aperture is important to understand.

Resolving Power

Resolving power = λ/(2 N.A.)

where N.A. = n • sinα

  α = angle formed from the outer ray of light admitted by the objective and optical axis

  α = A/2

Useful Magnification

The useful magnification of a light microscope is approximately 1000 times the numerical aperture of its objective. Generally the range of magnification used is between 40 and 400x. Calculating the maximum useful magnification for each objective used requires multiplying the figures printed on the objective by 1000 and comparing the result to the magnification.

Figure 17.

Photomicrograph

of Pigment

Distribution in

Red Human Hair

Figure 18.

Photomicrograph

of Pigment

Distribution in

Animal Hair

Ovoid bodies are large (larger than pigment granules), solid structures that are spherical to oval in shape, with very regular margins. They are abundant in some cattle (Figure 19) and dog (Figure 20) hairs as well as in other animal hairs. To varying degrees, they are also found in human hairs (Figure 21).

Figure 19.

Photomicrograph

of Ovoid Bodies

in Cattle Hair

Figure 20.

Photomicrograph

of Ovoid Bodies

in Dog Hair

Figure 21.

Photomicrograph

of Ovoid Bodies

in Human Hair

Hair Identification

Animal Versus Human Hairs

Human hairs are distinguishable from hairs of other mammals. Animal hairs are classified into the following three basic types.

 Guard hairs that form the outer coat of an animal and provide protection Fur or wool hairs that form the inner coat of an animal and provide insulationTactile hairs (whiskers) that are found on the head of animals provide sensory functions

Other types of hairs found on animals include tail hair and mane hair (horse). Human hair is not so differentiated and might be described as a modified combination of the characteristics of guard hairs and fur hairs.

Human hairs are generally consistent in color and pigmentation throughout the length of the hair shaft, whereas animal hairs may exhibit radical color changes in a short distance, called banding. The distribution and density of pigment in animal hairs can also be identifiable features. The pigmentation of human hairs is evenly distributed, or slightly more dense toward the cuticle, whereas the pigmentation of animal hairs is more centrally distributed, although more dense toward the medulla.

The medulla, when present in human hairs, is amorphous in appearance, and the width is generally less than one-third the overall diameter of the hair shaft. The medulla in animal hairs is normally continuous and structured and generally occupies an area of greater than one-third the overall diameter of the hair shaft.

The root of human hairs is commonly club-shaped (Figure 22), whereas the roots of animal hairs are highly variable.

Figure 22.

Photomicrograph

of Human

Hair Root

The scale pattern of the cuticle in human hairs is routinely imbricate. Animal hairs exhibit more variable scale patterns. The shape of the hair shaft is also more variable in animal hairs.

Human Hair Classifications

Hair evidence examined under a microscope provides investigators with valuable information. Hairs found on a knife or club may support a murder and/or assault weapon claim. A questioned hair specimen can be compared microscopically with hairs from a known individual, when the characteristics are compared side-by-side.

Human hairs can be classified by racial origin such as Caucasian (European origin), Negroid (African origin), and Mongoloid (Asian origin). In some instances, the racial characteristics exhibited are not clearly defined, indicating the hair may be of mixed-racial origin.

The region of the body where a hair originated can be determined with considerable accuracy by its gross appearance and microscopic characteristics. The length and color can be determined. It can also be determined whether the hair was forcibly removed, damaged by burning or crushing, or artificially treated by dyeing or bleaching.

The characteristics and their variations allow an experienced examiner to distinguish between hairs from different individuals. Hair examinations and comparisons, with the aid of a comparison microscope, can be valuable in an investigation of a crime.

DNA Examinations 

Hairs that have been matched or associated through a microscopic examination should also be examined by mtDNA sequencing. Although it is uncommon to find hairs from two different individuals exhibiting the same microscopic characteristics, it can occur. For this reason, the hairs or portions of the hairs should be forwarded for mtDNA sequencing. The combined procedures add credibility to each.

Although nuclear DNA analysis of hairs may provide an identity match, the microscopic examination should not be disregarded. The time and costs associated with DNA analyses warrant a preliminary microscopic examination. Often it is not possible to extract DNA fully, or there is not enough tissue present to conduct an examination. Hairs with large roots and tissue are promising sources of nuclear DNA. However, DNA examinations destroy hairs, eliminating the possibility of further microscopic examination.