Biological Evidence

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3 Trace Evidence I: Hairs and Fibers

LEARNING OBJECTIVES

After studying this chapter, you should be able to:

· • Recognize and understand the cuticle, cortex, and medulla areas of hair.

· • List the three phases of hair growth.

· • Appreciate the distinction between animal and human hairs.

· • List hair features that are useful for microscopic comparisons of human hairs.

· • Explain the proper collection of forensic hair evidence.

· • Describe and understand the role of DNA typing in hair comparisons.

· • Understand the differences between natural and manufactured fibers.

· • List the properties of fibers that are most useful for forensic comparisons.

· • Describe the proper collection of fiber evidence.

JEFFREY MACDONALD: FATAL VISION

The grisly murder scene that confronted police on February 17, 1970, is one that would not be wiped from memory. Summoned to the Fort Bragg residence of Captain Jeffrey MacDonald, a physician, police found the bludgeoned body of MacDonald’s wife. She had been repeatedly knifed, and her face was smashed to a pulp. MacDonald’s two children, ages 2 and 5, had been brutally and repeatedly knifed and battered to death.

Suspicion quickly fell on MacDonald. To the eyes of investigators, the murder scene had a staged appearance. MacDonald described a frantic effort to subdue four intruders who had slashed at him with an ice pick. However, the confrontation left MacDonald with minor wounds and no apparent defensive wounds on his arms. MacDonald then described how he had covered his slashed wife with his blue pajama top. Interestingly, when the body was removed, blue threads were observed under the body. In fact, blue threads matching the pajama top turned up throughout the house—nineteen in one child’s bedroom, including one beneath her fingernail, and two in the other child’s bedroom. Eighty-one blue fibers were recovered from the master bedroom, and two were located on a bloodstained piece of wood outside the house.

Forensic examination showed that the forty-eight ice pick holes in the pajama top were smooth and cylindrical, a sign that the top was stationary when it was slashed. Also, folding the pajama top demonstrated that the forty-eight holes actually could have been made by twenty-one thrusts of an ice pick. This coincided with the number of wounds that MacDonald’s wife sustained. As described in the book Fatal Vision, which chronicles the murder investigation, when MacDonald was confronted with adulterous conduct, he replied, “You guys are more thorough than I thought.” MacDonald is currently serving three consecutive life sentences.

The trace evidence transferred between individuals and objects during the commission of a crime, if recovered, often corroborates other evidence developed during the course of an investigation. Although in most cases physical evidence cannot by itself positively identify a suspect, laboratory examination may narrow the origin of such evidence to a group that includes the suspect. Using many of the instruments and techniques we have already examined, the crime laboratory has developed a variety of procedures for comparing and tracing the origins of physical evidence. This chapter will focus on the value of hairs and fibers as physical evidence.

Forensic Examination of Hair

Hair is encountered as physical evidence in a wide variety of crimes. However, any review of the forensic aspects of hair examination must start with the observation that it is not yet possible to individualize a human hair to any single head or body through its morphology, or structural characteristics. Over the years, criminalists have tried to isolate the physical and chemical properties of hair that could serve as individual characteristics of identity. Partial success has finally been achieved by isolating and characterizing the DNA present in hair.

The importance of hair as physical evidence cannot be overemphasized. Its removal from the body often denotes physical contact between a victim and perpetrator and hence a crime of a serious or violent nature. When hair is properly collected at the crime scene and submitted to the laboratory along with enough standard/reference samples, it can provide strong corroborative evidence for placing an individual at a crime site. The first step in the forensic examination of hair logically starts with its color and structure (i.e., morphology) and, if warranted, progresses to the more detailed DNA extraction, isolation, and characterization.

MORPHOLOGY OF HAIR

Hair is an appendage of the skin that grows out of an organ known as the hair follicle. The length of a hair extends from its root, or bulb, which is embedded in the follicle, continues into the shaft, and terminates at the tip. The shaft, which is composed of three layers—the cuticle , cortex , and medulla —is most intensely examined by the forensic scientist (see Figure 13-1 ).

cuticle

The scale structure covering the exterior of the hair.

cortex

The main body of the hair shaft.

medulla

A cellular column running through the center of the hair.

CUTICLE

Two features that make hair a good subject for establishing individual identity are its resistance to chemical decomposition and its ability to retain structural features over a long period of time. Much of this resistance and stability is attributed to the cuticle, a scale structure covering the exterior of the hair. The cuticle is formed by overlapping scales that always point toward the tip end of each hair. The scales form from specialized cells that have hardened (i.e., keratinized) and flattened in progressing from the follicle. There are three basic patterns that describe the appearance of the cuticle: cornal, spinous, and imbricate (see Figure 13-2 ).

Although the scale pattern is not a useful characteristic for individualizing human hair, the variety of patterns formed by animal hair makes it an important feature for species identification. Figure 13-3 shows the scale patterns of some animal hairs and of a human hair as viewed with a scanning electron microscope. Another method of studying the scale pattern of hair is to make a cast of its surface. This is done by embedding the hair in a soft medium, such as clear nail polish or softened vinyl. When the medium has hardened, the hair is removed, leaving a clear, distinct impression of the hair’s cuticle, ideal for examination with a compound microscope.

FIGURE 13-1 A cross-section of skin showing hair growing out of a tubelike structure called the follicle.

FIGURE 13-2 (a) The coronal, or crownlike, scale pattern resembles a stack of paper cups. (b) Spinous or petal-like scales are triangular in shape and protrude from the hair shaft. (c) The imbricate, or flattened-scale, type consists of overlapping scales with narrow margins. Richard Saferstein, Ph.D.

CORTEX

Contained within the protective layer of the cuticle is the cortex, the main body of the hair shaft. The cortex is made up of spindle-shaped cortical cells aligned in a regular array, parallel to the length of the hair. The cortex derives its major forensic importance from the fact that it is embedded with the pigment granules that give hair its color. The color, shape, and distribution of these granules provide important points of comparison among the hairs of different individuals.

FIGURE 13-3 Scale patterns of various types of hair: (a) human head hair (600×), (b) dog (1350×), (c) deer (120×), (d) rabbit (300×), (e) cat (2000×), and (f) horse (450×).

The structural features of the cortex are examined microscopically after the hair has been mounted in a liquid medium with a refractive index close to that of the hair. Under these conditions, the amount of light reflected off the hair’s surface is minimized, and the amount of light penetrating the hair is optimized.

MEDULLA

The medulla is a collection of cells that looks like a central canal running through a hair. In many animals, this canal is a predominant feature, occupying more than half of the hair’s diameter. The medullary index measures the diameter of the medulla relative to the diameter of the hair shaft and is normally expressed as a fraction. For humans, the index is generally less than one-third; for most other animals, the index is one-half or greater.

The presence and appearance of the medulla vary from individual to individual and even among the hairs of a given individual. Not all hairs have medullae, and when they do exist, the degree of medullation can vary. In this respect, medullae may be classified as being either continuous, interrupted, fragmented, or absent (see Figure 13-4 ). Human head hairs generally exhibit no medullae or have fragmented ones; they rarely show continuous medullation. One noted exception is in people of the Mongoloid race, who usually have head hairs with continuous medullae. Also, most animals have medullae that are either continuous or interrupted.

FIGURE 13-4 Medulla patterns.

FIGURE 13-5 Medulla patterns for various types of hair: (a) human head hair (400×), (b) dog (400×), (c) deer (500×), (d) rabbit (450×), (e) cat (400×), and (f) mouse (500×).

Another interesting feature of the medulla is its shape. Humans, as well as many animals, have medullae that give a nearly cylindrical appearance. Other animals exhibit medullae that have a patterned shape. For example, the medulla of a cat can best be described as resembling a string of pearls, whereas members of the deer family show a medullary structure consisting of spherical cells occupying the entire hair shaft. Figure 13-5 illustrates medullary sizes and forms for a number of common animal hairs and a human head hair.

A searchable database on CD-ROM of the thirty-five most common animal hairs encountered in forensic casework is commercially available. 1 This database allows an examiner to rapidly search for animal hairs based on scale patterns and/or medulla type using a PC. A typical screen presentation arising from such a data search is shown in Figure 13-6 .

ROOT

The root and other surrounding cells within the hair follicle provide the tools necessary to produce hair and continue its growth. Human head hair grows in three developmental stages, and the shape and size of the hair root is determined by the hair’s current growth phase. The three phases of hair growth are the anagen , catagen , and telogen phases .

anagen phase

The initial growth phase during which the hair follicle actively produces hair.

catagen phase

A transition stage between the anagen and telogen phases of hair growth.

telogen phase

The final growth phase in which hair naturally falls out of the skin.

FIGURE 13-6 Information on rabbit hair contained within the Forensic Animal Hair Atlas.

Courtesy RJ Lee Group, Inc. Monroeville, PA

FIGURE 13-7 Hair roots in the (a) anagen phase, (b) catagen phase, and (c) telogen phase (100×).

Courtesy Charles A. Linch

In the anagen phase (the initial growth phase), which may last up to six years, the root is attached to the follicle for continued growth, giving the root bulb a flame-shaped appearance ( Figure 13-7[a] ). When pulled from the root, some hairs in the anagen phase have a follicular tag . With the advent of DNA analysis, this follicular tag is important for individualizing hair.

follicular tag

A translucent piece of tissue surrounding the hair’s shaft near the root that contains the richest source of DNA associated with hair.

Hair continues to grow, but at a decreasing rate, during the catagen phase, which can last anywhere from two to three weeks. In the catagen phase, roots typically take on an elongated appearance ( Figure 13-7[b] ) as the root bulb shrinks and is pushed out of the hair follicle. Once hair growth ends, the telogen phase begins and the root takes on a club-shaped appearance ( Figure 13-7[c] ). Over two to six months, the hair is pushed out of the follicle, causing the hair to be naturally shed.

IDENTIFICATION AND COMPARISON OF HAIR

Most often the prime purpose for examining hair evidence in a crime laboratory is either to establish whether the hair is human or animal in origin or to determine whether human hair retrieved at a crime scene compares with hair from a particular individual. A careful microscopic examination of hair reveals morphological features that can distinguish human hair from animal hair. The hair of various animals also differs enough in structure that the examiner can often identify the species. Before reaching such a conclusion, however, the examiner must have access to a comprehensive collection of reference standards and the accumulated experience of hundreds of prior hair examinations. Scale structure, medullary index, and medullary shape are particularly important in hair identification.

The most common request when hair is used as forensic evidence is to determine whether hair recovered at the crime scene compares to hair removed from a suspect. In most cases, such a comparison relates to hair obtained from the scalp or pubic area. Ultimately, the evidential value of the comparison depends on the degree of probability with which the examiner can associate the hair in question with a particular individual.

FACTORS IN COMPARISON OF HAIR

Although animal hair normally can be distinguished from human hair with little difficulty, human hair comparisons must be undertaken with extreme caution. Hair tends to exhibit variable morphological characteristics, not only from one person to another but also within a single individual. In comparing hair, the criminalist is particularly interested in matching color, length, and diameter. Other important features are the presence or absence of a medulla and the distribution, shape, and color intensity of the pigment granules in the cortex. A microscopic examination may also distinguish dyed or bleached hair from natural hair. A dyed color is often present in the cuticle as well as throughout the cortex. Bleaching, on the other hand, tends to remove pigment from the hair and gives it a yellowish tint.

If hair has grown since it was last bleached or dyed, the natural-end portion will be quite distinct in color. An estimate of the time since dyeing or bleaching can be made because hair grows approximately 1 centimeter per month. Other significant but less frequent features may be observed in hair. For example, morphological abnormalities may be present as a result of certain diseases or nutrient deficiencies. Also, the presence of fungal and nit infections can further link a hair specimen to a particular individual.

MICROSCOPIC EXAMINATION OF HAIR

A comparison microscope is an invaluable tool that allows the examiner to view the questioned and known hair together, side by side. Any variations in the microscopic characteristics will thus be readily observed. Because hair from any part of the body exhibits a range of characteristics, it is necessary to have an adequate number of known hairs that are representative of all its features when making a comparison.

Although the microscopic comparison of hairs has long been accepted as an appropriate approach for including and excluding questioned hairs against standard/reference hairs, many forensic scientists have long recognized that this approach is very subjective and is highly dependent on the skills and integrity of the analyst, as well as the hair morphology being examined. However, until the advent of DNA analysis, the forensic science community had no choice but to rely on the microscope to carry out hair comparisons.

Any lingering doubts about the necessity of augmenting microscopic hair examinations with DNA analysis evaporated with the publication of an FBI study describing significant error rates associated with microscopic comparison of hairs. 2 Hair evidence submitted to the FBI for DNA analysis between 1996 and 2000 was examined both microscopically and by DNA analysis.

Approximately 11 percent of the hairs (nine out of eighty) in which FBI hair examiners found a positive microscopic match between questioned and standard/reference hairs were found to be nonmatches when they were later subjected to DNA analysis. The course of events is clear: Microscopic hair comparisons must be regarded by police and courts as presumptive in nature, and all positive microscopic hair comparisons must be confirmed by DNA determinations.

QUESTIONS ABOUT HAIR EXAMINATION

A number of questions may be asked to further ascertain the present status of forensic hair examinations. The answers to these questions can be of great significance to the investigator working with hair evidence.

Can the Body Area from Which a Hair Originated Be Determined?

Normally it is easy to determine the body area from which a hair came. For example, scalp hairs generally show little diameter variation and have a more uniform distribution of pigment when compared to other body hairs. Pubic hairs are short and curly, with wide variations in shaft diameter, and usually have continuous medullae. Beard hairs are coarse, are normally triangular in cross-section, and have blunt tips acquired from cutting or shaving.

Can the Racial Origin of Hair Be Determined?

In many instances, the examiner can distinguish hair originating from members of different races; this is especially true of Caucasian and Negroid head hair. Negroid hairs are normally kinky, containing dense, unevenly distributed pigments. Caucasian hairs are usually straight or wavy, with very fine to coarse pigments that are more evenly distributed when compared to Negroid hair. Mongoloid hairs often have a dense pigment distribution, but they normally don’t exhibit the pigment clumping seen in Negroid hairs. Mongoloids also tend to have thicker hair shaft diameters when compared to the other two races.

Sometimes a cross-sectional examination of hair may help identify race. Cross-sections of hair from Caucasians are oval to round in shape, Mongoloid generally exhibit a round cross-sectional shape, and cross-sections of Negroid hair are flat to oval in shape. However, all of these observations are general, with many possible exceptions. The criminalist must approach the determination of race from hair with caution and a good deal of experience.

Can the Age and Sex of an Individual Be Determined from a Hair Sample?

The age of an individual cannot be learned from a hair examination with any degree of certainty except in the case of infant hairs, which are fine and short and have fine pigmentation. Although the presence of dye or bleach on the hair may offer some clue to sex, present hairstyles make these characteristics less valuable than they were in the past. The recovery of nuclear DNA either from tissue adhering to a hair or from the root structure of the hair will allow a determination of whether the hair originated from a male or female.

Is It Possible to Determine Whether Hair Was Forcibly Removed from the Body?

A microscopic examination of the hair root may establish whether the hair fell out or was pulled out of the skin. A hair root with follicular tissue (root sheath cells) adhering to it, as shown in Figure 13-8 , indicates a hair that has been pulled out either by a person or by brushing or combing. Hair naturally falling off the body has a bulbous-shaped root free of any adhering tissue.

The absence of sheath cells cannot always be relied on for correctly judging whether hair has been forcibly pulled from the body. In some cases the root of a hair is devoid of any adhering tissue even when it has been pulled from the body. Apparently, an important consideration is how quickly the hair is pulled out of the head. Hairs pulled quickly from the head are much more likely to have sheath cells compared to hairs that have been removed slowly from the scalp. 3

CASE FILES CENTRAL PARK JOGGER CASE REVISITED

On April 19, 1989, a young woman left her apartment around nine p.m. to jog in New York’s Central Park. Nearly five hours later, she was found comatose lying in a puddle of mud in the park. She had been raped, her skull was fractured, and she had lost 75 percent of her blood. When the woman recovered, she had no memory of what happened to her. The brutality of the crime sent shock waves through the city and seemed to fuel a national perception that crime was running rampant and unchecked through the streets of New York.

Already in custody at the station house of the Central Park Precinct was a group of 14- and 15-year-old boys who had been rounded up leaving the park earlier in the night by police who suspected that they had been involved in a series of random attacks. Over the next two days, four of the teenagers gave videotaped statements, which they later recanted, admitting to participating in the attack. Ultimately, five of the teenagers were charged with the crime.

Interestingly, none of the semen collected from the victim could be linked to any of the defendants. However, according to the testimony of a forensic analyst, two head hairs collected from the clothing of one of the defendants microscopically compared to those of the victim, and a third hair collected from the same defendant’s T-shirt microscopically compared to the victim’s pubic hair. Besides these three hairs, a fourth hair was found to be microscopically similar to the victim’s. This hair was recovered from the clothing of Steven Lopez, who was originally charged with rape but not prosecuted for the crime.

Hairs were the only pieces of physical evidence offered by the district attorney to directly link any of the teenagers to the crime. The hairs were cited by the district attorney as proof for the jury that the videotaped confessions of the teenagers were reliable. The five defendants were convicted and ultimately served from nine to thirteen years.

In August 1989, more than three months after the jogger attack, New York police arrested a man named Matias Reyes, who pleaded guilty to murdering a pregnant woman, raping three other women, and committing a robbery. For these crimes Reyes was sentenced to thirty-three years to life. In January 2002, Reyes also confessed to the Central Park attack. Follow-up tests revealed that Reyes’s DNA compared to semen recovered from the jogger’s body and her sock. Other DNA tests showed that the hairs offered into evidence at the original trial did not come from the victim and so could not be used to link the teenagers to the crime as the district attorney had argued. After an eleven-month reinvestigation of the original charges, a New York State Supreme Court judge dismissed all the convictions against the five teenage suspects in the Central Park jogger case.

Courtesy AP Wide World Photos

FIGURE 13-8 Forcibly removed head hair with follicular tissue attached.

Are Efforts Being Made to Individualize Human Hair?

As we will see in Chapter 15 , forensic scientists routinely isolate and characterize individual variations in DNA. Forensic hair examiners can link human hair to a particular individual by characterizing the nuclear DNA in the hair root or in follicular tissue adhering to the root (see Figure 13-8 ). Recall that the follicular tag is the richest source of DNA associated with hair. In the absence of follicular tissue, an examiner must extract DNA from the hair root.

nuclear DNA

DNA that is present in the nucleus of a cell and that is inherited from both parents.

The growth phase of hair is a useful predictor of the likelihood of successfully typing DNA in human hair. 4 Examiners have a higher success rate in extracting DNA from hair roots in the anagen phase or from anagen-phase hairs entering the catagen phase of growth. Telogen-phase hairs have an inadequate amount of DNA for typing. Because most hairs are naturally shed and are expected to be in the telogen stage, these observations do not portend well for hairs collected at crime scenes. However, some crime scenes are populated with forcibly removed hairs that are expected to be rich sources for nuclear DNA.

When a questioned hair does not have adhering tissue or a root structure amenable to isolation of nuclear DNA, there is an alternative source of information: mitochondrial DNA . Unlike the nuclear DNA described earlier, which is located in the nuclei of practically every cell in the body, mitochondrial DNA is found in cellular material outside the nucleus. Interestingly, unlike nuclear DNA, which is passed down from both parents, mitochondrial DNA is transmitted only from mother to child. Importantly, many more copies of mitochondrial DNA than nuclear DNA are located in the cells. For this reason, the success rate of finding and typing mitochondrial DNA is much greater from samples that have limited quantities of nuclear DNA, such as hair. Hairs 1 to 2 centimeters long can be subjected to mitochondrial analysis with extremely high odds of success. This subject is discussed in greater detail in Chapter 15 .

mitochondrial DNA

DNA present in small structures (i.e., mitochondria) outside the nucleus of a cell. Mitochondria supply energy to the cell. This form of DNA is inherited maternally (from the mother).

Can DNA Individualize a Human Hair?

In some cases, the answer is yes. As we will learn in Chapter 15 , nuclear DNA produces frequencies of occurrence as low as one in billions or trillions. On the other hand, mitochondrial DNA cannot individualize human hair. However, its diversity within the human population often permits the exclusion of a significant portion of a population as potential contributors of a hair sample. Ideally, the combination of a positive microscopic comparison and an association through nuclear or mitochondrial DNA analysis strongly links a questioned hair and standard/reference hairs. However, a word of caution: Mitochondrial DNA cannot distinguish microscopically similar hairs from individuals who are maternally related.

COLLECTION AND PRESERVATION OF HAIR EVIDENCE

When questioned hairs are submitted to a forensic laboratory for examination, they must always be accompanied by an adequate number of standard/reference samples from the victim of the crime and from individuals suspected of having deposited hair at the crime scene. We have learned that hair from different parts of the body varies significantly in its physical characteristics. Likewise, hair from any one area of the body can also have a wide range of characteristics. For this reason, the questioned and standard/reference hairs must come from the same area of the body; one cannot, for instance, compare head hair to pubic hair. It is also important that the collection of standard/ reference hair be carried out in a way that ensures a representative sampling of hair from any one area of the body.

CASE FILES

The murder of Ennis Cosby, son of entertainer Bill Cosby, at first appeared unsolvable. It was a random act. When his car tire went flat, Ennis pulled off the road and called a friend on his cellular phone to ask for assistance. Shortly thereafter, an assailant demanded money and, when Cosby didn’t respond quickly enough, shot him once in the temple. Acting on a tip from a friend of the assailant, police investigators later found a .38-caliber revolver wrapped in a blue cap miles from the crime scene. Mikail Markhasev was arrested and charged with murder.

Bill Cosby and his son Ennis Cosby.

Courtesy Andrea Mohin, The New York Times

At the trial, the district attorney introduced firearms evidence to show that the recovered gun had fired the bullet that killed Cosby. A single hair also recovered from the hat dramatically linked Markhasev to the crime: Los Angeles Police Department forensic analyst Harry Klann identified six DNA markers from the follicular tissue adhering to the hair root that matched Markhasev’s DNA. This particular DNA profile is found in 1 out of 15,500 members of the general population. On hearing all the evidence, the jury deliberated and convicted Markhasev of murder.

Forensic hair comparisons generally involve either head hair or pubic hair. Collecting fifty full-length hairs from all areas of the scalp normally ensures a representative sampling of head hair. Likewise, a minimum collection of twenty-four full-length pubic hairs should cover the range of characteristics present in this type of hair. In rape cases, care must first be taken to comb the pubic area with a clean comb to remove all loose foreign hair present before the victim is sampled for standard/reference hair. The comb should then be packaged in a separate envelope.

Because a hair may vary in color and other morphological features over its entire length, the entire hair is collected. This requirement is best accomplished by either pulling the hair out of the skin or clipping it at the skin line. During an autopsy, hair samples are routinely collected from victims of suspicious deaths. Because the autopsy may occur early in an investigation, the need for hair standard/reference samples may not always be apparent. However, one should never rule out the possible involvement of hair evidence in subsequent investigative findings. Failure to make this simple collection may result in complicated legal problems later.

Quick Review

· • The hair shaft is composed of three layers called the cuticle, cortex, and medulla and is the part of a hair most intensely examined by the forensic scientist.

· • When comparing strands of hair, the criminalist is particularly interested in matching the color, length, and diameter. Other important features for comparing hair are the presence or absence of a medulla and the distribution, shape, and color intensity of pigment granules in the cortex.

· • The likelihood of successfully detecting DNA in hair roots is higher in hair being examined in its anagen or early growth phase than in its catagen or telogen phases.

· • The follicular tag, a translucent piece of tissue surrounding the hair’s shaft near the root, is a rich source of DNA associated with hair. Mitochondrial DNA can also be extracted from the hair shaft.

· • All positive microscopic hair comparisons must be confirmed by DNA analysis.

Forensic Examination of Fibers

Just as hair left at a crime scene can be used for identification, so can the fibers that compose fabrics and garments. Fibers may become important evidence in incidents that involve personal contact—such as homicide, assault, and sexual offenses—in which cross-transfers may occur between the clothing of suspect and victim. Similarly, the force of impact between a hit-and-run victim and a vehicle often leaves fibers, threads, or even whole pieces of clothing adhering to parts of the vehicle. Fibers may also become fixed in screens or on glass that is broken in the course of a breaking-and-entering attempt.

Regardless of where and under what conditions fibers are recovered, their ultimate value as forensic evidence depends on the criminalist’s ability to narrow their origin to a limited number of sources or even to a single source. Unfortunately, mass production of garments and fabrics has limited the value of fiber evidence in this respect, and only rarely do fibers recovered at a crime scene provide individual identification with a high degree of certainty.

TYPES OF FIBERS

For centuries, humans depended on fibers derived from natural sources such as plants and animals. However, early in the twentieth century, the first manufactured fiber—rayon—became a practical reality, followed in the 1920s by the introduction of cellulose acetate. Since the late 1930s, scientists have produced dozens of new fibers. In fact, there have been greater advances in the development of fibers, fabrics, finishes, and other textile-processing techniques since 1900 than in the preceding five thousand years of recorded history. Today, such varied items as clothing, carpeting, drapes, wigs, and even artificial turf attest to the predominant role that manufactured fibers have come to play in our culture and environment. When discussing forensic examination of fibers, it is convenient to classify them into two broad groups: natural and manufactured.

NATURAL FIBERS

Natural fibers are wholly derived from animal or plant sources. Natural fibers encountered in crime laboratory examinations come primarily from animals. These include hair coverings from such animals as sheep (wool), goats (mohair, cashmere), camels, fiamas, alpacas, and vicuñas. Fur fibers include those obtained from animals such as mink, rabbit, beaver, and muskrat.

natural fibers

Fibers derived entirely from animal or plant sources.

The forensic examination of animal fibers uses the same procedures discussed in the previous section for the forensic examination of animal hairs. The identification and comparison of such fibers relies solely on a microscopic examination of color and morphological characteristics. Again, a sufficient number of standard/reference specimens must be examined to establish the range of fiber characteristics that make up the suspect fabric.

FIGURE 13-9 Photomicrograph of cottonfiber (450×).

By far the most prevalent plant fiber is cotton. The wide use of undyed white cotton fibers in clothing and other fabrics has made its evidential value almost meaningless, but the presence of dyed cotton in a combination of colors has, in some cases, enhanced its evidential significance. The microscopic view of cotton fiber shown in Figure 13-9 reveals its most distinguishing feature—its ribbonlike shape with twists at irregular intervals.

MANUFACTURED FIBERS

Beginning with the introduction of rayon in 1911 and the development of nylon in 1939, manufactured fibers have increasingly replaced natural fibers in garments and fabrics. Such fibers are marketed under hundreds of trade names. To reduce consumer confusion, the US Federal Trade Commission has approved “generic” or family names for the grouping of all manufactured fibers. Many of these generic classes are produced by several manufacturers and are sold under a confusing variety of trade names. For example, in the United States, polyesters are marketed under names that include Dacron, Fortrel, and Kodel. In England, polyesters are called Terylene. Table 13.1 lists major generic fibers, along with common trade names and their characteristics and applications.

manufactured fibers

Fibers derived from either natural or synthetic polymers.

The first machine-made fibers were manufactured from raw materials derived from cotton or wood pulp, and these are still being made. The raw materials are processed, and pure cellulose is extracted from them. Depending on the type of fiber desired, the cellulose may be chemically treated and dissolved in an appropriate solvent before it is forced through the small holes of a spinning jet, or spinneret, to produce the fiber. Fibers manufactured from natural raw materials in this manner are classified as regenerated fibers and commonly include rayon, acetate, and triacetate, all of which are produced from regenerated cellulose.

Most of the fibers currently manufactured are produced solely from synthetic chemicals and are therefore classified as synthetic fibers. These include nylons, polyesters, and acrylics. The creation of synthetic fibers became a reality only when scientists developed a method of synthesizing long-chained molecules called polymers.

In 1930, chemists discovered an unusual characteristic of one of the polymers under investigation. When a glass rod in contact with viscous material in a beaker was slowly pulled away, the substance adhered to the rod and formed a fine filament that hardened as soon as it entered the cool air. Furthermore, the cold filaments could be stretched several times their extended length to produce a flexible, strong, and attractive fiber. This first synthetic fiber was improved and then marketed as nylon. Since then, fiber chemists have successfully synthesized new polymers and have developed more efficient methods for manufacturing them. These efforts have produced a multitude of synthetic fibers.

IDENTIFICATION AND COMPARISON OF MANUFACTURED FIBERS

The evidential value of fibers lies in the criminalist’s ability to trace their origin. Obviously, if the examiner is presented with fabrics that can be exactly fitted together at their torn edges, the fabrics must be of common origin.

More often, however, the criminalist obtains a limited number of fibers for identification and comparison. Generally, in these situations obtaining a physical match is unlikely, and the examiner must resort to a side-by-side comparison of the standard/reference and crime-scene fibers.

MICROSCOPIC EXAMINATION OF FIBERS

The first and most important step in the examination is a microscopic comparison for color and diameter using a comparison microscope. Unless these two characteristics agree, there is little reason to suspect a match. Other morphological features that may aid in the comparison are lengthwise striations (lined markings) on the surface of some fibers and the pitting of the fiber’s surface with delustering particles (usually titanium dioxide) added in the manufacturing process to reduce shine (see Figure 13-10 ).

TABLE 13.1 Major Generic Fibers

MAJOR GENERIC FIBER	CHARACTERISTICS	MAJOR DOMESTIC AND INDUSTRIAL USES
Acetate	· • Luxurious feel and appearance · • Wide range of colors and lusters · • Excellent drapability and softness · • Relatively fast-drying · • Shrink-, moth-, and mildew-resistant	Apparel: Blouses, dresses, foundation garments, lingerie, linings, shirts, slacks, sportswear Fabrics: Brocade, crepe, double knits, faille, knitted jerseys, lace, satin, taffeta, tricot Home Furnishings: Draperies, upholstery Other: Cigarette filters, fiberfill for pillows, quilted products
Acrylic	· • Soft and warm · • Wool-like · • Retainsshape · • Resilient · • Quick-drying · • Resistanttomoths, sunlight, oil, and chemicals	Apparel: Dresses, infant wear, knitted garments, skiwear, socks, sportswear, sweaters Fabrics: Fleece and pile fabrics, face fabrics in bonded fabrics, simulated furs, jerseys Home Furnishings: Blankets, carpets, draperies, upholstery Other: Auto tops, awnings, hand-knitting and craft yarns, industrial and geotextile fabrics
Aramid	· • Does not melt · • Highly flame-resistant · • Great strength · • Great resistance to stretch · • Maintains shape and form at high temperatures	Hot-gas filtration fabrics, protective clothing, military helmets, protective vests, structural composites for aircraft and boats, sailcloth, tires, ropes and cables, mechanical rubber goods, marine and sporting goods
Bicomponent	· • Thermal bonding · • Self-bulking · • Very fine fibers · • Unique cross-sections · • The functionality of special polymers or additives at reduced cost	Uniform distribution of adhesive; fiber remains a part of structure and adds integrity; customized sheath materials to bond various materials; wide range of bonding temperatures; cleaner, environmentally friendly (no effluent); recyclable; lamination/molding/densification of composites
Lyocell	· • Soft, strong, absorbent · • Good dyeability · • Fibrillates during wet processing to produce special textures	Dresses, slacks, and coats
Melamine	· • White and dyeable · • Flame resistance and low thermal conductivity · • High-heat dimensional stability · • Processable on standard textile equipment	Fire-Blocking Fabrics: Aircraft seating, fire blockers for upholstered furniture in high-risk occupancies (e.g., to meet California TB 133 requirements) Protective Clothing: Firefighters’ turnout gear, insulating thermal liners, knit hoods, molten metal splash apparel, heat-resistant gloves Filter Media: High-capacity, high-efficiency, high-temperature baghouse air filters
Modacrylic	· • Soft · • Resilient · • Abrasion- and flame-resistant · • Quick-drying · • Resists acids and alkalies · • Retains shape	Apparel: Deep-pile coats, trims, linings, simulated fur, wigs and hairpieces Fabrics: Fleece fabrics, industrial fabrics, knit-pile fabric backings, nonwoven fabrics Home Furnishings: Awnings, blankets, carpets, flame-resistant draperies and curtains, scatter rugs Other: Filters, paint rollers, stuffed toys
Nylon	· • Exceptionally strong · • Supple · • Abrasion-resistant · • Lustrous · • Easy to wash · • Resists damage from oil and many chemicals · • Resilient · • Low in moisture absorbency	Apparel: Blouses, dresses, foundation garments, hosiery, lingerie and underwear, raincoats, ski and snow apparel, suits, windbreakers Home Furnishings: Bedspreads, carpets, draperies, curtains, upholstery Other: Air hoses, conveyor and seat belts, parachutes, racket strings, ropes and nets, sleeping bags, tarpaulins, tents, thread, tire cord, geotextiles
Olefin	· • Unique wicking properties that make it very comfortable · • Abrasion-resistant · • Quick-drying · • Resistant to deterioration from chemicals, mildew, perspiration, rot, and weather · • Sensitive to heat · • Soil-resistant · • Strong; very lightweight · • Excellent colorfastness	Apparel: Pantyhose, underwear, knitted sports shirts, men’s half-hose, men’s knitted sportswear, sweaters Home Furnishings: Carpet and carpet backing, slipcovers, upholstery Other: Dye nets, filter fabrics, laundry bags, sandbags, geotextiles, automotive interiors, cordage, doll hair, industrial sewing thread
Polyester	· • Strong · • Resistant to stretching and shrinking · • Resistant to most chemicals · • Quick-drying · • Crisp and resilient when wet or dry · • Wrinkle- and abrasion-resistant · • Retains heat-set pleats and creases · • Easy to wash	Apparel: Blouses, shirts, career apparel, children’s wear, dresses, half-hose, insulated garments, ties, lingerie and underwear, permanent press garments, slacks, suits Home Furnishings: Carpets, curtains, draperies, sheets and pillowcases Other: Fiberfill for various products, fire hoses, power belting, ropes and nets, tire cord, sail, V-belts
PBI	· • Extremely flame-resistant · • Outstanding comfort factor combined with thermal and chemical stability properties · • Will not burn or melt · • Low shrinkage when exposed to flame	Suitable for high-performance protective apparel such as firefighters’ turnout coats, astronaut space suits, and applications in which fire resistance is important
Rayon	· • Highly absorbent · • Soft and comfortable · • Easy to dye · • Versatile · • Good drapability	Apparel: Blouses, coats, dresses, jackets, lingerie, linings, millinery, rainwear, slacks, sports shirts, sportswear, suits, ties, work clothes Home Furnishings: Bedspreads, blankets, carpets, curtains, draperies, sheets, slipcovers, tablecloths, upholstery Other: Industrial products, medical-surgical products, nonwoven products, tire cord
Spandex	· • Can be stretched 500 percent without breaking · • Can be stretched repeatedly and recover original length · • Lightweight · • Stronger and more durable than rubber · • Resistant to body oils	Apparel (articles in which stretch is desired): Athletic apparel, bathing suits, delicate laces, foundation garments, golf jackets, ski pants, slacks, support and surgical hose

Source: American Fiber Manufacturers Assoc. Inc., Washington, DC, www.fingersource.com Reprinted by permission.

FIGURE 13-10 Photomicrographs of synthetic fibers: (a) cellulose triacetate (450×) and (b) olefin fiber embedded with titanium dioxide particles (450×).

FIGURE 13-11 Cross-sectional shapes of fibers.

The cross-sectional shape of a fiber may also help characterize the fiber (see Figure 13-11 ). 5 In the early 1880s, Wayne Williams was charged and tried for the murder of two individuals in the Atlanta, Georgia, region. During the eight-week trial, evidence linking Williams to those murders and to the murder of ten other individuals was introduced. An essential part of the government’s case was the numerous fibers linking Williams to the murders. Unusually shaped yellow-green fibers discovered on a number of the murder victims were linked to a carpet in the Williams home. This fiber was a key element in proving Williams’s guilt. A photomicrograph of this unusually shaped fiber is shown in Figure 13-12 .

FIGURE 13-12 A scanning electron photomicrograph of the cross-section of a nylon fiber removed from a sheet used to transport the body of a murder victim. The fiber, associated with a carpet in Wayne Williams’s home, was manufactured in 1971 in relatively small quantities.

Courtesy Federal Bureau of Investigation, Washington, DC

Although two fibers may seem to have the same color when viewed under the microscope, compositional differences may actually exist in the dyes that were applied to them during their manufacture. In fact, most textile fibers are impregnated with a mixture of dyes selected to obtain a desired shade or color. The significance of a fiber comparison is enhanced when the forensic examiner can show that the questioned and standard/reference fibers have the same dye composition.

ANALYTICAL TECHNIQUES USED IN FIBER EXAMINATION

In Chapter 11 , we saw how a chemist can use selective absorption of light by materials to characterize them. In particular, light in the ultraviolet, visible, and infrared regions of the electromagnetic spectrum is most helpful for this purpose. Unfortunately, in the past, forensic chemists were unable to take full advantage of the capabilities of spectrophotometry for examining trace evidence because most spectrophotometers are not well suited for examining the very small particles frequently encountered as evidence. Recently, linking the microscope to a computerized spectrophotometer has added a new dimension to its capability. This combination has given rise to a new instrument called the microspectrophotometer. In many respects, this is an ideal marriage from the forensic scientist’s viewpoint.

The visible-light microspectrophotometer is a convenient way for analysts to compare the colors of fibers through spectral patterns. This technique is not limited by sample size; a fiber as small as 1 millimeter long or less can be examined by this type of microscope. The examination is nondestructive and is carried out on fibers simply mounted on a microscope slide.

CHEMICAL COMPOSITION

Before the forensic scientist can reach a conclusion that two or more fibers compare, it must be shown that the fibers in question have the same chemical composition. In this respect, tests are performed to confirm that all of the fibers involved belong to the same broad generic class. Additionally, the comparison will be substantially enhanced if it can be demonstrated that all of the fibers belong to the same subclassification within their generic class. For example, at least four types of nylon are available in commercial and consumer markets, including nylon 6, nylon 6-10, nylon 11, and nylon 6-6. Although all types of nylon have many properties in common, each may differ in physical shape, appearance, and dyeability because of modifications in their basic chemical structure.

CLOSER ANALYSIS THE MICROSPECTROPHOTOMETER

With the development of the microspectrophotometer, a forensic analyst can view a particle under a microscope while a beam of light is directed at the particle to obtain its absorption spectrum. Depending on the type of light employed, an examiner can acquire either a visible or an infrared (IR) spectral pattern of the substance being viewed under the microscope. The obvious advantage of this approach is that it provides added information to characterize trace quantities of evidence. A microspectrophotometer designed to measure the uptake of visible light by materials is shown here.

Visual comparison of color is usually one of the first steps in examining paint, fiber, and ink evidence. Such comparisons are easily obtained using a comparison microscope. A forensic scientist can use the microspectrophotometer to compare the color of materials visually while plotting an absorption spectrum for each item under examination. This displays the exact wavelengths at which each item absorbs in the visible-light spectrum. Occasionally, colors that appear similar by visual examination show significant differences in their absorption spectra.

Another emerging technique in forensic science is the use of the IR microspectrophotometer to examine fibers and paints. The “fingerprint” IR spectrum (see Figure 1 and 2 in the Case File on page 338 ) is unique for each chemical substance. Therefore, obtaining such a spectrum from either a fiber or a paint chip allows the analyst to better identify and compare the type of chemicals from which these materials are manufactured. With a microspectrophotometer, a forensic analyst can view a substance through the microscope and at the same time have the instrument plot the infrared absorption spectrum for that material.

A visible-light microspectrophotometer.

Courtesy CRAIC Technologies Inc., Altadena, CA, www.microspectra.com

Textile chemists have devised numerous tests for determining the class of a fiber. However, unlike the textile chemist, the criminalist frequently does not have the luxury of a substantial quantity of the fabric to work with and must therefore select tests that will yield the most information with the least amount of material. Only a single fiber may be available for analysis, and often this may amount to no more than a minute strand recovered, for example, from a fingernail scraping from a homicide or rape victim.

INFRARED ABSORPTION

The polymers that compose a manufactured fiber, like any organic substance, selectively absorb infrared light in a characteristic pattern. Infrared spectrophotometry thus provides a rapid and reliable method for identifying the generic class, and in some cases the subclass, of a fiber. The infrared microspectrophotometer combines a microscope with an infrared spectrophotometer. Such a combination makes possible the infrared analysis of a small, single-strand fiber while it is being viewed under a microscope.

SIGNIFICANCE OF FIBER EVIDENCE

Once a fiber match has been determined, the question of the significance of such a finding is bound to be raised. In reality, no analytical technique permits the criminalist to link a fiber strand definitively to any single garment. Furthermore, except in the most unusual circumstances, no statistical databases are available for determining the probability of a fiber’s origin. Considering the mass distribution of synthetic fibers and the constantly changing fashion tastes of our society, it is highly unlikely that such data will be available in the foreseeable future.

Despite these limitations, an investigator should not discount or minimize the significance of a fiber association. An enormous variety of fibers exists in our society. By simply looking at the random individuals we meet every day, we can see how unlikely it is to find two people wearing identically colored fabrics (with the exception of blue denims or white cottons). There are thousands of different-colored fibers in our environment. Combine this with the fact that forensic scientists compare not only the color of fibers but also their size, shape, microscopic appearance, chemical composition, and dye content, and one can now begin to appreciate how unlikely it is to find two indistinguishable colored fibers on two randomly selected sources.

Furthermore, the significance of a fiber association increases dramatically when the analyst can link two or more distinctly different fibers to the same object. Likewise, the associative value of fiber evidence is dramatically enhanced if it is accompanied by other types of physical evidence linking a person or object to a crime. As with most class evidence, the significance of a fiber comparison is dictated by the circumstances of the case; by the location, number, and nature of the fibers examined; and, most important, by the judgment of an experienced examiner.

Collection and Preservation of Fiber Evidence

As criminal investigators have become more aware of the potential contribution of trace physical evidence to the success of their investigations, they have placed greater emphasis on conducting thorough crime-scene searches for evidence of forensic value. Their skill and determination at carrying out these tasks is tested in the collection of fiber-related evidence. Fiber evidence can be associated with virtually any type of crime. It usually cannot be seen with the naked eye and thus can be easily overlooked by someone not specifically searching for it.

An investigator committed to optimizing the laboratory’s chances for locating minute strands of fibers identifies and preserves potential “carriers” of fiber evidence. Relevant articles of clothing should be packaged carefully in paper bags. Each article must be placed in a separate bag to avoid cross-contamination of evidence. Scrupulous care must be taken to prevent articles of clothing from different people or from different locations from coming into contact. Such articles must not even be placed on the same surface prior to packaging. Likewise, carpets, rugs, and bedding are to be folded carefully to protect areas suspected of containing fibers. Car seats should be carefully covered with polyethylene sheets to protect fiber evidence, and knife blades should be covered to protect adhering fibers. If a body is thought to have been wrapped at one time in a blanket or carpet, adhesive tape lifts of exposed body areas may reveal fiber strands.

Occasionally the field investigator may need to remove a fiber from an object, particularly if loosely adhering fibrous material may be lost in transit to the laboratory. These fibers must be removed with a clean forceps and placed in a small sheet of paper, which, after folding and labeling, should be placed inside another container. Again, scrupulous care must be taken to prevent contact between fibers collected from different objects or from different locations.

In the laboratory, the search for fiber evidence on clothing and other relevant objects, as well as in debris, is time consuming and tedious and will test the skill and patience of the examiner. The crime-scene investigator can manage this task by collecting only relevant items for examination—pinpointing areas where a likely transfer of fiber evidence occurred and then ensuring the proper collection and preservation of these materials.

CASE FILES FATAL VISION REVISITED

Dr. Jeffrey MacDonald, pictured here, was convicted in 1979 of murdering his wife and two young daughters. The events surrounding the crime and the subsequent trial were recounted in Joe McGinniss’s best-selling book Fatal Vision. The focus of Dr. MacDonald’s defense was that intruders entered his home and committed these violent acts. Eleven years after this conviction, Dr. MacDonald’s attorneys filed a petition for a new trial, claiming the existence of “critical” new evidence.

The defense asserted that wig fibers found on a hairbrush in the MacDonald residence were evidence that an intruder dressed in a wig entered the MacDonald home on the day of the murder. Subsequent examination of this claim by the FBI Laboratory focused on a blond fall (a type of artificial hair extension) frequently worn by Dr. MacDonald’s wife. Fibers removed from the fall were shown to clearly match fibers on the hairbrush. The examination included the use of infrared microspectrophotometry to demonstrate that the suspect wig fibers were chemically identical to fibers found in the composition of Mrs. MacDonald’s fall (see Figure 1 ). Hence, although wig fibers were found at the crime scene, the source of these fibers could be accounted for: Mrs. MacDonald’s fall.

Jeffrey MacDonald in 1995 at Sheridan, Oregon, Federal Correctional Institution.

Courtesy AP Wide World Photos

Another piece of evidence cited by Dr. MacDonald’s lawyers was a bluish-black woolen fiber found on the body of Mrs. MacDonald. They claimed that this fiber compared to a bluish-black woolen fiber recovered from the club used to assault her. These wool fibers were central to Dr. MacDonald’s defense that the “intruders” wore dark-colored clothing. Initial examination showed that the fibers were microscopically indistinguishable. However, the FBI also compared the two wool fibers by visible-light microspectrophotometry. Comparison of their spectra clearly showed that their dye compositions differed, providing no evidence of outside intruders (see Figure 2 ). Ultimately, the US Supreme Court denied the merits of Jeffrey MacDonald’s petition for a new trial.

Source: Based on information contained in B. M. Murtagh and M. P. Malone, “Fatal Vision Revisited,” Police Chief (June 1993): 15.

FIGURE 1 A fiber comparison made with an infrared spectrophotometer. The infrared spectrum of a fiber from Mrs. MacDonald’s fall compares to a fiber recovered from a hairbrush in the MacDonald home. These fibers were identified as modacrylics, the most common type of synthetic fiber used in the manufacture of human hair goods.

Courtesy SA Michael Malone, FBI Laboratory, Washington, DC

FIGURE 2 The visible-light spectrum for the woolen fiber recovered from Mrs. MacDonald’s body is clearly different from that of the fiber recovered from the club used to assault her.

Courtesy SA Michael Malone, FBI Laboratory, Washington, DC

Quick Review

· • Fibers may be classified into two broad groups: natural and manufactured.

· • Most fibers currently manufactured are produced solely from synthetic chemicals and are therefore classified as synthetic fibers. They include nylons, polyesters, and acrylics.

· • Microscopic comparisons between questioned and standard/reference fibers are initially undertaken for color and diameter characteristics. Other features that could be important in comparing fibers are striations on the surface of the fiber, the presence of delustering particles, and the cross-sectional shape of the fiber.

· • Using a visible-light microspectrophotometer is a convenient way for analysts to compare the colors of fibers through spectral patterns.

· • Infrared microspectrophotometry is a reliable method for identifying the chemical composition of fibers.

· • Fiber evidence collected at each location should be placed in separate containers to avoid cross-contamination. Care must be taken to prevent articles of clothing from different people or from different locations from coming into contact with each other.

VIRTUAL LAB Forensic Hair Analysis

To perform a virtual forensic hair analysis, go to www.pearsoncustom.com/us/vlm/

VIRTUAL LAB Examination of Textile Fibers by Microscopy

To perform a virtual fiber examination lab, go to www.pearsoncustom.com/us/vlm/

CHAPTER REVIEW

· • The hair shaft is composed of three layers called the cuticle, cortex, and medulla and is the part of the hair most intensely examined by the forensic scientist.

· • The likelihood of successfully detecting DNA in hair roots is higher in hair being examined in its anagen or early growth phase that in its catagen or telogen phases.

· • All positive microscopic hair comparisons must be confirmed by DNA analysis.

· • Fibers may be classified into two broad groups: natural and manufactured.

· • Most fibers currently manufactured are produced solely from synthetic chemicals and are therefore classified as synthetic fibers. They include nylons, polyesters, and acrylics.

· • Microscopic comparisons between questioned and standard/ reference fibers are initially undertaken for color and diameter characteristics. Other features that could be important in comparing fibers are striations on the surface of the fiber, the presence of delustering particles, and the cross-sectional shape of the fiber.

· • Using a visible-light microspectrophotometer is a convenient way for analysts to compare the colors of fibers through spectral patterns.

· • Infrared microspectrophotometry is a reliable method for identifying the chemical composition of fibers.

KEY TERMS

anagen phase 323

catagen phase 323

cortex 320

cuticle 320

follicular tag 324

manufactured fibers 331

medulla 320

mitochondrial DNA 328

natural fibers 330

nuclear DNA 328

telogen phase 323

REVIEW QUESTIONS

Hair is an appendage of the skin, growing out of an organ known as the _____________.

The three layers of the hair shaft are the _____________, the _____________, and the _____________.

The scale pattern of hair’s _____________can be observed by making a cast of its surface in clear nail polish or softened vinyl.

The _____________contains the pigment granules that impart color to hair.

The central canal running through many hairs is known as the _____________.

The diameter of the medulla relative to the diameter of the hair shaft is the _____________.

Human hair generally has a medullary index of less than _____________; the hair of most animals has an index of _____________or greater.

True or False: Human head hairs generally exhibit no medullae. _____________

True or False: If a medulla exhibits a pattern, the hair is animal in origin. _____________

10.

The three stages of hair growth are the _____________, _____________, and _____________phases.

11.

True or False: Individual hairs can show variable morphological characteristics within a single individual. _____________

12.

True or False: A single hair cannot be individualized to one person by microscopic examination. _____________

13.

In making hair comparisons, it is best to view the hairs side by side under a(n) _____________microscope.

14.

_____________hairs are short and curly, with wide variation in shaft diameter.

15.

True or False: It is possible to estimate when hair was last bleached or dyed by microscopic examination. _____________

16.

True or False: The age and sex of the individual from whom a hair sample has been taken can be determined through an examination of the hair’s morphological features. _____________

17.

True or False: Hair forcibly removed from the body sometimes has follicular tissue adhering to its root. _____________

18.

Microscopic hair comparisons must be regarded by police and courts as presumptive in nature, and all positive microscopic hair comparisons must be confirmed by _____________ typing.

19.

A hair root in the _____________or _____________ growth phase is a likely candidate for DNA typing.

20.

A minimum collection of _____________full-length hairs normally ensures a representative sampling of head hair.

21.

A minimum collection of _____________full-length pubic hairs is recommended to cover the range of characteristics present in this region of the body.

22.

The ultimate value of fibers as forensic evidence depends on the ability to narrow their _____________to a limited number of sources or even to a single source.

23.

_____________fibers are derived totally from animal or plant sources.

24.

The most prevalent natural plant fiber is _____________.

25.

_____________fibers such as rayon, acetate, and triacetate are manufactured from natural raw materials such as cellulose.

26.

Fibers manufactured solely from synthetic chemicals are classified as _____________.

27.

True or False: Polyester was the first synthetic fiber. _____________

28.

True or False: A first step in the forensic examination of fibers is to compare color and diameter. _____________

29.

The microspectrophotometer employing _____________ light is a convenient way for analysts to compare the colors of fibers through spectral patterns.

30.

The microspectrophotometer employing _____________ light provides a rapid and reliable method for identifying the generic class of a single fiber.

31.

True or False: Statistical databases are available for determining the probability of a fiber’s origin. _____________

32.

True or False: Normally, fibers possess individual characteristics. _____________

33.

In order to preserve fiber evidence not originally apparent to the investigator, all _____________of possible fiber evidence should be carefully collected and packaged.

APPLICATION AND CRITICAL THINKING

Indicate the phase of growth of each of the following hairs:

· a)The root is club shaped.

· b)The hair has a follicular tag.

· c)The root bulb is flame shaped.

· d)The root is elongated.

A criminalist studying a dyed sample hair notices that the dyed color ends about 1.5 centimeters from the tip of the hair. Approximately how many weeks before the examination was the hair dyed? Explain your answer.

Following are descriptions of several hairs. Based on these descriptions, indicate the likely race of the person from whom the hair originated.

· a)Evenly distributed, fine pigmentation.

· b)Continuous medullation.

· c)Dense, uneven pigmentation.

· d)Wavy with a round cross-section.

Criminalist Pete Evett is collecting fiber evidence from a murder scene. He notices fibers on the victim’s shirt and trousers, so he places both of these items of clothing in a plastic bag. He also sees fibers on a sheet near the victim, so he balls up the sheet and places it in a separate plastic bag. Noticing fibers adhering to the windowsill from which the attacker gained entrance, Pete carefully removes it with his fingers and places it in a regular envelope. What mistakes, if any, did Pete make while collecting this evidence?

For each of the following human hair samples, indicate the medulla pattern present.

· (a)_____________

· (b)_____________

· (c)_____________

· (d)_____________

· (e)_____________

· (f)_____________

· (g)_____________

· (h)_____________

· (i)_____________

The most common scale patterns found on hairs are generally classified as coronal, spinous, and imbricate. Examine the scale casts of animal hairs shown here and indicate the scale pattern of each.

· (a)_____________

_____________

· (b)_____________

_____________

· (c)_____________

_____________

· (d)_____________

_____________

· (e)_____________

_____________

· (f)_____________

_____________

· (g)_____________

_____________

· (h)_____________

_____________

A young child is kidnapped from her school playground. Shown on the left is a reference sample of the kidnapped child’s hair. The only cars that left the parking lot before the child was discovered to be missing were those of four cafeteria workers. The car of each worker was searched and hairs collected. These recovered hairs are shown on the right. Which recovered hair, if any, is consistent with that of the victim and warrants further investigation?

ENDNOTES

CH.15

15 Biological Stain Analysis: DNA

Court POOL/ZUMA Press/Newscom

LEARNING OBJECTIVES

After studying this chapter, you should be able to:

· • List the A-B-O antigens and antibodies found in each of the four blood types: A, B, AB, and O.

· • List and describe forensic tests used to characterize a stain as blood.

· • List the laboratory tests necessary to characterize seminal stains.

· • Explain how to properly preserve suspect blood and semen stains for laboratory examination.

· • Contrast chromosomes and genes.

· • Name the parts of a nucleotide and explain how they are linked together to form DNA.

· • Understand the concept of base pairing as it relates to the double-helix structure of DNA.

· • Explain the technology of polymerase chain reaction (PCR) and how it applies to forensic DNA typing

· • Understand the DNA-typing technique known as short tandem repeats (STRs).

· • Describe the difference between nuclear and mitochondrial DNA.

· • Understand the use of computerized DNA databases in criminal investigation.

· • List the necessary procedures for the proper preservation of biological evidence for laboratory DNA analysis.

O. J. SIMPSON: A MOUNTAIN OF EVIDENCE

On June 12, 1994, police who arrived at the home of Nicole Simpson viewed a horrific scene. The bodies of O. J. Simpson’s estranged wife and her friend Ron Goldman were found on the path leading to the front door of Nicole’s home. Both bodies were covered in blood and had received deep knife wounds. Nicole’s head was nearly severed from her body. This was not a well-planned murder. A trail of blood led away from the murder scene. Blood was found in O. J. Simpson’s Bronco. There were blood drops on O. J.’s driveway and in the foyer of his home. A blood-soaked sock was located in O. J. Simpson’s bedroom, and a bloodstained glove rested on the ground outside his residence (see accompanying photo).

As DNA was extracted and profiled from each bloodstained article, a picture emerged that seemed to irrefutably link Simpson to the murders. A trail of DNA leaving the crime scene was consistent with O. J.’s profile, as was the DNA found in Simpson’s home. Simpson’s DNA profile was found in the Bronco along with that of both victims. The glove contained the DNA profiles of Nicole and Ron, and the sock had Nicole’s DNA profile. At trial, the defense team valiantly fought back. Miscues in evidence collection were craftily exploited. The defense strategy was to paint a picture of not only an incompetent investigation but one that was tinged with dishonest police planting evidence. The strategy worked. O. J. Simpson was acquitted of murder.

In 1901, Karl Landsteiner announced one of the most significant discoveries of the twentieth century—the typing of blood—a finding that earned him a Nobel Prize twenty-nine years later. For years physicians had attempted to transfuse blood from one individual to another. Their efforts often ended in failure because the transfused blood tended to coagulate, or clot, in the body of the recipient, causing instantaneous death. Landsteiner was the first to recognize that all human blood was not the same; instead, he found that blood is distinguishable by its group, or type.

Out of Landsteiner’s work came the classification system that we call the A-B-O system. Now physicians have the key for properly matching the blood of a donor to that of a recipient. Because one blood type cannot be mixed with a different blood type without disastrous consequences, this discovery, of course, had important implications for blood transfusion, and millions of lives have since been saved.

Meanwhile, Landsteiner’s findings opened a new field of research in the biological sciences. Others began to pursue the identification of additional characteristics that could further differentiate blood. By 1937, the Rh factor in blood had been demonstrated, and shortly thereafter, numerous blood factors or groups were discovered. More than one hundred blood factors have been identified. However, the ones in the A-B-O system are still the most important for properly matching a donor and recipient for a transfusion.

Until the early 1990s, forensic scientists focused on blood factors, such as A-B-O, as offering the best means for linking blood to an individual. What made these factors so attractive was that, in theory, no two individuals, except for identical twins, could be expected to have the same combination of blood factors. In other words, blood factors are controlled genetically and have the potential of being a highly distinctive feature for personal identification. What makes this observation so relevant is the great frequency of bloodstains at crime scenes, especially crimes of the most serious nature: homicides, assaults, and sexual assaults. Consider, for example, a transfer of blood between the victim and assailant during a struggle, that is, the transfer of a victim’s blood to the suspect’s garment, or vice versa. If the criminalist could individualize human blood by identifying all of its known factors, the result would be strong evidence for linking the suspect to the crime.

The advent of DNA technology has dramatically altered the approach of forensic scientists toward the individualization of bloodstains and other biological evidence. The search for genetically controlled blood factors in bloodstains has been abandoned in favor of characterizing biological evidence by select regions of our deoxyribonucleic acid (DNA) , which carries the body’s genetic information. As a result, the individuation of dried blood and other biological evidence has become a reality and has significantly altered the role that crime laboratories play in criminal investigations. In fact, the high sensitivity of DNA analysis and the resultant search for DNA evidence has even altered the types of materials collected from crime scenes.

deoxyribonucleic acid (DNA)

The molecules that carry the body’s genetic information.

The Nature of Blood

The word blood refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. The fluid portion of blood is called plasma ; it is composed principally of water and accounts for 55 percent of blood content. Suspended in the plasma are solid materials consisting chiefly of several types of cells: red blood cells (i.e., erythrocytes), white blood cells (i.e., leukocytes), and platelets. The solid portion of blood accounts for 45 percent of its content. Blood clots when a protein in the plasma known as fibrin traps and enmeshes the red blood cells. If the clotted material were removed from the blood, a pale yellowish liquid known as serum would be left.

plasma

The fluid portion of unclotte blood.

serum

The liquid that separates from the blood when a clot is formed.

Considering the complexity of blood, a full discussion of its function and chemistry would extend beyond the scope of this text. Instead, this chapter concentrates on the components of blood that are directly pertinent to the forensic aspects of blood identification: the red blood cells and the blood serum.

ANTIGENS AND ANTIBODIES

Red blood cells transport oxygen from the lungs to the body tissues and remove carbon dioxide from tissues by transporting it back to the lungs, where it is exhaled. However, for reasons unrelated to the red blood cell’s transporting mission, on the surface of each cell are millions of characteristic chemical structures called antigens . Antigens impart specific characteristics to the red blood cells. Blood antigens are grouped into systems depending on their relationship to one another. More than fifteen blood antigen systems have been identified to date; of these, the A-B-O and Rh systems are the most important.

antigen

A substance, usually a protein, that stimulates the body to produce antibodies against it.

If an individual has type A blood, this simply means that each red blood cell has A antigens on its surface; similarly, all type B individuals have B antigens, and the red blood cells of type AB individuals contain both A and B antigens. Type O individuals have neither A nor B antigens on their cells. Hence, the presence or absence of A and B antigens on the red blood cells determines a person’s blood type in the A-B-O system.

Another important blood antigen has been named the Rh factor, or D antigen. Those people who have the D antigen are said to be Rh positive; those without this antigen are Rh negative. In routine blood banking, the presence or absence of the three antigens—A, B, and D—must be tested to determine the compatibility of the donor and recipient.

Serum is important because it contains proteins known as antibodies . The fundamental principle of blood typing is that, for every antigen, there exists a specific antibody. Each antibody symbol contains the prefix anti-, followed by the name of the antigen for which it is specific. Hence, anti-A is specific only for the A antigen, anti-B for the B antigen, and anti-D for the D antigen. The antibody-containing serum is referred to as the antiserum , meaning a serum that reacts against something (i.e., antigens).

antibody

A protein in the blood serum that destroys or inactivates a specific antigen.

antiserum

Blood serum that contains specific antibodies.

An antibody reacts only with its specific antigen and no other. Thus, if serum containing anti-B is added to red blood cells carrying the B antigen, the two will combine, causing the antibody to attach itself to the cell. Antibodies are normally bivalent—that is, they have two reactive sites. This means that each antibody can simultaneously be attached to antigens located on two different red blood cells. This creates a vast network of cross-linked cells usually seen in the form of clumping, or agglutination (see Figure 15-1 ).

agglutination

The clumping together of red blood cells by the action of an antibody.

FIGURE 15-1 Agglutination of blood cells.

Let’s look a little more closely at this phenomenon. In normal blood, shown in Figure 15-2(a) , antigens on red blood cells and antibodies coexist without destroying each other because the antibodies present are not specific toward any of the antigens. However, suppose a foreign serum added to the blood introduces a new antibody. This results in a specific antigen–antibody reaction that immediately causes the red blood cells to link together, or agglutinate, as shown in Figure 15-2(b) .

FIGURE 15-2 (a) A microscopic view of normal red blood cells (500x). (b) A microscopic view of agglutinated red blood cells (500x).

Evidently, nature has taken this situation into account, for when we examine the serum of type A blood, we find anti-B but no anti-A. Similarly, type B blood contains only anti-A, type O blood has both anti-A and anti-B, and type AB blood contains neither anti-A nor anti-B. The antigen and antibody components of normal blood are summarized in the following table:

Blood Type	Antigens on Red Blood Cells	Antibodies in Serum
A	A	Anti-B
B	B	Anti-A
AB	AB	Neither anti-A nor anti-B
O	Neither A nor B	Both anti-A and anti-B

The reasons for the fatal consequences of mixing incompatible blood during a transfusion should now be quite obvious. For example, the transfusion of type A blood into a type B patient will cause the natural anti-A in the blood of the type B patient to react promptly with the incoming A antigens, resulting in agglutination. In addition, the incoming anti-B of the donor will react with the B antigens of the patient.

Immunoassay Techniques

The concept of a specific antigen-antibody reaction is being applied in other areas unrelated to blood typing. Most significant, similar reactions are being applied to the detection of drugs in blood and urine. Antibodies that react with drugs do not exist naturally; however, they can be produced in animals such as rabbits by first combining the drug with a protein and injecting this combination into the animal. This drug-protein complex acts as an antigen stimulating the animal to produce antibodies (see Figure 15-3 ). The recovered blood serum of the animal now contains antibodies that are specific or nearly specific to the drug.

FIGURE 15-3 Stimulating production of drug antibodies.

Currently, each day, thousands of individuals are voluntarily being subjected to urinalysis tests for the presence of commonly abused drugs. These individuals include military personnel, transportation industry employees, police and corrections personnel, and candidates undergoing preemployment drug screening. Immunoassay testing for drugs has proved quite suitable for handling the large volume of specimens that must be rapidly analyzed on a daily basis for drug content. Testing laboratories have available to them a variety of commercially prepared sera that were developed in animals injected with any one of a variety of drugs. Once a particular serum is added to a urine specimen, it’s designed to interact with either opiates, cannabinoids, amphetamines, phencyclidine, barbiturates, methadone, or another type of drug that might be present. A word of caution: Immunoassay is only presumptive in nature, and its result must be confirmed by additional testing.

Quick Review

· • An antibody reacts or agglutinates only with its specific antigen. The concept of specific antigen-antibody reactions has been applied to techniques for the detection of commonly abused drugs in blood and urine.

· • Every red blood cell contains either an A antigen, a B antigen, both antigens, or no antigen (this is called type O). The type of antigen on one’s red blood cells determines one’s A-B-O blood type. Persons with type A blood have A antigens on their red blood cells, those with type B blood have B antigens, those with type AB blood have both antigens, and those with type O blood have no antigens on their red blood cells.

· • To produce antibodies capable of reacting with drugs, a specific drug is combined with a protein, and this combination is injected into an animal such as a rabbit. This drug-protein complex acts as an antigen, stimulating the animal to produce antibodies. The recovered blood serum of the animal will now contain antibodies that are specific or nearly specific to the drug.

Forensic Characterization of Bloodstains

The criminalist must answer the following questions when examining dried blood: (1) Is it blood? (2) From what species did the blood originate? (3) If the blood is human, how closely can it be associated with a particular individual?

COLOR TESTS

The determination that a substance is blood is best made by means of a preliminary color test. For many years, the most common test was the benzidine color test. However, because benzidine has been identified as a known carcinogen, its use has generally been discontinued, and the chemical phenolphthalein is usually substituted (this test is also known as the Kastle-Meyer color test).

Both the benzidine and Kastle-Meyer color tests are based on the observation that blood hemoglobin possesses peroxidase-like activity. Peroxi-dases are enzymes that accelerate the oxidation of several classes of organic compounds when combined with peroxides. For example, when a bloodstain, phenolphthalein reagent, and hydrogen peroxide are mixed together, oxidation of the hemoglobin in the blood produces a deep pink color.

The Kastle-Meyer test is not a specific test for blood; some vegetable materials, for instance, may turn Kastle-Meyer pink. These substances include potatoes and horseradish. However, such materials will probably not be encountered in criminal situations, and thus, from a practical point of view, a positive Kastle-Meyer test is highly indicative of blood. Field investigators also have found Hemastix strips a useful presumptive field test for blood. Designed as a urine dipstick test for blood, the strip can be moistened with distilled water and placed in contact with a suspect bloodstain. The appearance of a green color indicates the presence of blood.

WebExtra 15.1

See a Color Test for Blood www.mycrimekit.com

LUMINOL AND BLUESTAR

Another important presumptive identification test for blood is the luminol test. 1 Unlike the benzidine and Kastle-Meyer tests, the reaction of luminol with blood produces light rather than color. After spraying luminol reagent onto suspect items, agents darken the room; any bloodstains produce a faint blue glow, known as luminescence. Using luminol, investigators can quickly screen large areas for bloodstains. A relatively new product, Bluestar ( www.bluestar-forensic.com ), is now available to be used in place of luminol. Bluestar is easy to mix in the field. Its reaction with blood can be observed readily without having to create complete darkness.

The luminol and Bluestar tests are extremely sensitive—capable of detecting bloodstains diluted up to 100,000 times. For this reason, spraying large areas such as carpets, walls, flooring, or the interior of a vehicle may reveal blood traces or patterns that would have gone unnoticed under normal lighting conditions (see Figure 15-4 ). Luminol and Bluestar will not interfere with any subsequent DNA testing. 2

FIGURE 15-4 (a) A section of a linoleum floor photographed under normal light. This floor was located in the residence of a missing person. (b) The same section of the floor shown in (a) after spraying with luminol. A circular pattern was revealed. Investigators concluded that the circular blood pattern was left by the bottom of a bucket used during cleanup of the blood. A small clump of sponge, blood, and hair was found near where this photograph was taken.

Courtesy North Carolina State Bureau of Investigation

MICROCRYSTALLINE TESTS

The identification of blood can be made more specific if microcrystalline tests are performed on the material. Several tests are available; the two most popular ones are the Takayama and Teichmann tests. Both depend on the addition of specific chemicals to the blood to form characteristic crystals containing hemoglobin derivatives. Crystal tests are far less sensitive than color tests for blood identification and are more susceptible to interference from contaminants that may be present in the stain.

PRECIPITIN TEST

Once the stain has been characterized as blood, the serologist determines whether the blood is of human or animal origin. The standard test for this is the precipitin test. Precipitin tests are based on the fact that when animals (usually rabbits) are injected with human blood, antibodies form that react with the invading human blood to neutralize its presence. The investigator can recover these antibodies by bleeding the animal and isolating the blood serum, which contains antibodies that specifically react with human antigens. For this reason, the serum is known as human antiserum. In the same manner, by injecting rabbits with the blood of other known animals, virtually any kind of animal antiserum can be produced. Antiserums are commercially available for human blood and for the blood of a variety of commonly encountered animals, such as dogs, cats, and deer.

Several techniques have been devised for performing precipitin tests on bloodstains. The classic method is to layer an extract of the bloodstain on top of the human antiserum in a capillary tube. Human blood—or, for that matter, any protein of human origin in the extract—reacts specifically with antibodies present in the antiserum, indicated by the formation of a cloudy ring or band at the interface of the two liquids (see Figure 15-5 ).

FIGURE 15-5 The precipitin test.

GEL DIFFUSION

Another precipitin method, called gel diffusion, takes advantage of the fact that antibodies and antigens diffuse or move toward one another on a plate coated with a gel medium made from a natural polymer called agar. The extracted bloodstain and the human antiserum are placed in separate holes opposite each other on the gel. If the blood is human, a line of precipitation forms where the antigens and antibodies meet.

Similarly, the antigens and antibodies can be induced to move toward one another under the influence of an electrical field. In the electrophoretic method, an electrical potential is applied to the gel medium; a specific antigen–antibody reaction is denoted by a line of precipitation formed between the hole containing the blood extract and the hole containing the human antiserum (see Figure 15-6 ).

The precipitin test is very sensitive and requires only a small amount of blood for testing. Human bloodstains that have been dry for ten to fifteen years and longer may still give a positive precipitin reaction. Even extracts of tissue from mummies four to five thousand years old have given positive reactions with this test. Furthermore, human bloodstains diluted by washing in water and left with only a faint color may still yield a positive precipitin reaction (see Figure 15-7 ).

Once it has been determined that the bloodstain is human, an effort must be made to associate the stain with or disassociate the stain from a particular individual. Until the mid-1990s, routine characterization of bloodstains included the determination of A-B-O types; however, the widespread use of DNA profiling, or typing, has relegated this subject to one of historical interest only.

FIGURE 15-6 Gel diffusion.

FIGURE 15-7 Results of the precipitin test of dilutions of human serum up to 1 in 4,096 against a human antiserum. A reaction is visible for blood dilutions up to 1 in 256.

Courtesy Millipore Biomedica, Acton, MA

Quick Review

· • The criminalist must be prepared to answer the following questions when examining dried blood: (1) Is it blood? (2) From what species did the blood originate? (3) If the blood is of human origin, how closely can it be associated to a particular individual?

· • The determination that a substance is blood is best made by means of a preliminary color test. A positive result from the Kastle-Meyer color test is highly indicative of blood.

· • The luminol and Bluestar tests are used to search out trace amounts of blood located at crime scenes.

· • The precipitin test uses antisera, normally derived from rabbits that have been injected with the blood of a known animal, to determine the species origin of a questioned bloodstain.

Forensic Characterization of Semen

Many cases encountered in a forensic laboratory involve sexual offenses, making it necessary to examine evidence for the presence of seminal stains. The forensic examination of articles for seminal stains can be considered a two-step process. First, before any tests can be conducted, the stain must be located. Considering the potential number and soiled condition of outer garments, undergarments, and possibly bed clothing submitted for examination, this can be an arduous task. Once located, stains must be subjected to tests that will prove their identity. A stain may even be tested for the blood type of the individual from whom it originated.

TESTING FOR SEMINAL STAINS

Often seminal stains are visible on a fabric because they exhibit a stiff, crusty appearance. However, reliance on such appearance for locating the stain is unreliable and is useful only when the stain is in an obvious area. If the fabric has been washed or contains only minute quantities of semen, visual examination offers little chance of detecting the stain. The best way to locate and at the same time characterize a seminal stain is to perform the acid phosphatase color test.

ACID PHOSPHATASE TEST

Acid phosphatase is an enzyme that is secreted by the prostate gland into seminal fluid. Its concentrations in seminal fluid are up to four hundred times those found in any other body fluid. Its presence can easily be detected when it comes into contact with an acidic solution of sodium alpha naphthylphosphate and Fast Blue B dye. Also, 4-methylumbelliferyl phosphate (MUP) will fluoresce (i.e., emit light) under UV light when it comes into contact with acid phosphatase.

acid phosphatase

An enzyme found in high concentrations in semen.

The utility of the acid phosphatase test is apparent when it becomes necessary to search many garments or large pieces of fabric for seminal stains. Simply moistening a filter paper with water and rubbing it lightly over the suspect area transfers any acid phosphatase present to the filter paper. Placing a drop or two of the sodium alpha naphthylphosphate and Fast Blue B solution on the paper produces a purple color that indicates the acid phosphatase enzyme. In this manner, any fabric or surface can be systematically searched for seminal stains.

If it is necessary to search extremely large areas—for example, a bedsheet or carpet—the article can be tested in sections, narrowing the location of the stain with each successive test. Alternatively, the garment can be pressed against a suitably sized piece of moistened filter paper. The paper is then sprayed with MUP solution. Semen stains appear as strongly fluorescent areas under UV light. A negative reaction can be interpreted as an absence of semen. Although some vegetable and fruit juices (such as cauliflower and watermelon), fungi, contraceptive creams, and vaginal secretions give a positive response to the acid phosphatase test, none of these substances normally reacts with the speed of seminal fluid. A reaction time of less than 30 seconds is considered a strong indication of semen.

MICROSCOPIC EXAMINATION OF SEMEN

Semen can be unequivocally identified by the presence of spermatozoa. When spermatozoa are located through a microscopic examination, the stain is definitely identified as having been derived from semen. Spermatozoa are slender, elongated structures 50 to 70 microns long, each with a head and a thin flagellate tail (see Figure 15-8 ). The criminalist can normally locate them by immersing the stained material in a small volume of water. Rapidly stirring the liquid transfers a small percentage of the spermatozoa present into the water. A drop of the water is dried onto a microscope slide, then stained and examined under a compound microscope at a magnification of approximately 400×.

Considering the extremely large number of spermatozoa found in seminal fluid (the normal male releases 250 to 600 million spermatozoa during ejaculation), the chance of locating one should be very good; however, this is not always true. One reason is that spermatozoa bind tightly to cloth materials. 3 Also, spermatozoa are extremely brittle when dry and easily disintegrate if the stain is washed or when the stain is rubbed against another object, as happens frequently in the handling and packaging of this type of evidence. Furthermore, sexual crimes may involve males who have an abnormally low sperm count, a condition known as oligospermia , or who have no spermatozoa at all in their seminal fluid ( aspermia ). Significantly, aspermatic individuals are increasing in numbers because of the growing popularity of vasectomies.

oligospermia

An abnormally low sperm count.

aspermia

The absence of sperm; sterility in males.

FIGURE 15-8 A photomicrograph of human spermatozoa (300×).

John Walsh\Photo Researchers, Inc.

PROSTATE-SPECIFIC ANTIGEN (PSA)

Analysts often examine stains or swabs that they suspect contain semen (because of the presence of acid phosphatase), but that yield no detectable spermatozoa. How, then, can one reliably prove the presence of semen? The solution to this problem came with the discovery in the 1970s of a protein called p30 or prostate-specific antigen (PSA). At first, this protein was thought to be prostate specific and hence a unique identifier of semen. However, additional research has shown that low levels of p30 may be detectable in other human tissues. A more reasonable approach to the unequivocal identification of semen is to use a positive p30 test in combination with an acid phosphatase color test with a reaction time of less than 30 seconds. 4

When p30 is isolated and injected into a rabbit, it stimulates the production of polyclonal antibodies (anti-p30). The serum collected from these immunized rabbits can then be used to test suspected semen stains. As shown in Figure 15-9 , the stain extract is placed in one well of an electrophoretic plate and the anti-p30 in an opposite well. When an electric potential is applied, the antigens and antibodies move toward each other. The formation of a visible line midway between the two wells shows the presence of p30 in the stain and indicates that the stain originated from semen.

FIGURE 15-9 PSA testing by electrophoresis.

FIGURE 15-10 An antibody–antigen–antibody “sandwich,” or complex, is seen as a colored band arising from the attached blue dye. This signifies the presence of PSA in the extract of a stain and positively identifies human semen.

A more elegant approach to identifying PSA (or p30) is shown in Figure 15-10 . First, a monoclonal PSA antibody is attached to a dye and placed on a porous membrane. Monoclonal antibodies are specially designed to attack a single antigen site. Next, an extract from a sample suspected of containing PSA is placed on the membrane. If PSA is present in the extract, it combines with the monoclonal PSA antibody to form a PSA antigen–monoclonal PSA antibody complex. This complex migrates along the membrane, where it interacts with a PSA antibody imbedded in the membrane. The antibody–antigen–antibody “sandwich” that forms is apparent by the presence of a colored line (see Figure 15-10 ). This monoclonal antibody technique is about 100 times as sensitive as the electrophoretic method for detecting PSA.

Once the material is proved to be semen, the next task is to associate the semen as closely as possible with an individual. As we will learn, forensic scientists can link seminal material to one individual with DNA technology. Just as important is the fact that this technology can exonerate many of those wrongfully accused of sexual assault.

Quick Review

· • The best way to locate and characterize a seminal stain is to perform the acid phosphatase color test.

· • The presence of spermatozoa is a unique identifier of semen. Also, the protein called prostate-specific antigen (PSA), also known as p30, is useful in combination with the acid phosphatase color test for characterizing a sample stain as semen.

· • Forensic scientists can link seminal material to an individual by DNA typing.

Collection of Sexual Assault Evidence

Seminal constituents on a sexual assault victim are important evidence that sexual intercourse has taken place, but their absence does not necessarily mean that a sexual assault did not occur. Physical injuries such as bruises and bleeding tend to confirm that a violent assault occurred. Furthermore, the forceful physical contact between victim and assailant may result in a transfer of physical evidence such as blood, semen, hairs, and fibers. The presence of such evidence helps forge a vital link in the chain of circumstances surrounding a sexual crime.

To protect this kind of evidence, all the outer garments and undergarments from the victim should be carefully removed and packaged separately in paper (not plastic) bags. A clean bedsheet should be placed on the floor and a clean paper sheet placed over it. The victim must remove her shoes before standing on the paper. The person should disrobe while standing on the paper in order to collect any loose foreign material falling from the clothing. Each piece of clothing should be collected as it is removed and placed in a separate paper bag to avoid cross-contamination. The paper sheet should be folded carefully so that all foreign materials are contained inside. If appropriate, bedding or the object on which the assault took place should be submitted to the laboratory for processing.

Items suspected of containing seminal stains must be handled carefully. Folding an article at the location of a stain may cause it to flake off, as will rubbing the stained area against the surface of the packaging material. If, under unusual circumstances, it is not possible to transport the stained article to the laboratory, the stained area should be cut out and submitted along with a separately packaged unstained piece as a substrate control.

In the laboratory, analysts try to link seminal material to a source using DNA typing. Because an investigator may transfer his or her DNA types to a stain through perspiration, stained articles must be handled with care, minimizing direct personal contact. The evidence collector must wear disposable latex gloves when such evidence must be touched.

The sexual assault victim must undergo a medical examination as soon as possible after the assault. At this time, the appropriate items of physical evidence are collected by trained personnel. Evidence collectors should have an evidence-collection kit from the local crime laboratory (see Figure 15-11 ).

The following procedure should be followed by a medical professional to collect items of physical evidence from the sexual assault victim:

· 1. Pubic combings . Place a paper towel under the buttocks and comb the pubic area for loose or foreign hairs.

· 2. Pubic hair standard/reference samples . Cut fifteen to twenty full-length hairs from the pubic area at the skin line.

· 3. External genital dry-skin areas . Swab with at least one dry swab and one moistening swab.

· 4. Vaginal swabs and smear . Using two swabs simultaneously, carefully swab the vaginal area and let the swabs air-dry before packaging. Using two additional swabs, repeat the swabbing procedure and smear the swabs onto separate microscope slides, allowing them to air-dry before packaging.

FIGURE 15-11(left) A victim sexual assault evidence collection kit showing the kit envelope, kit instructions, medical history and assault information forms, and a foreign materials collection bag.

Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com

FIGURE 15-11(right) A victim sexual assault evidence collection kit showing collection bags for outer clothing, underpants, debris, pubic hair combings, pubic hair standard/reference samples, vaginal swabs, and rectal swabs.

Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com

FIGURE 15-11A victim sexual assault evidence collection kit showing collection bags for oral swabs and smear, standard/reference head hairs, saliva sample, and blood samples, and anatomical drawings.

Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com

· 5. Cervix swabs . Using two swabs simultaneously, carefully swab the cervix area and let the swabs air-dry before packaging.

· 6. Rectal swabs and smear . To be taken when warranted by case history. Using two swabs simultaneously, swab the rectal canal, smearing one of the swabs onto a microscope slide. Allow both samples to air-dry before packaging.

· 7. Oral swabs and smear . To be taken if oral–genital contact occurred. Use two swabs simultaneously to swab the cheek area and gum line. Using both swabs, prepare one smear slide. Allow both swabs and the smear to air-dry before packaging.

· 8. Head hairs . Cut at the skin line a minimum of ten full-length hairs from each of the following scalp locations: center, front, back, left side, and right side. A total of at least fifty hairs should be cut and submitted to the laboratory.

· 9. Blood sample . Collect at least 7 milliliters in a vacuum tube containing the preservative EDTA. (The blood sample can be used for DNA typing as well as for toxicological analysis if required.)

· 10. Fingernail scrapings . Scrape the undersurface of the nails with a dull object over a piece of clean paper to collect debris. Use separate paper, one for each hand.

· 11. All clothing . Package as described earlier.

· 12. Urine specimen . Collect 30 milliliters or more of urine from the victim for analysis for Rohypnol, GHB, and other substances associated with drug-facilitated sexual assaults.

Often, during the investigation of a sexual assault, the victim reports that a perpetrator engaged in biting, sucking, or licking areas of the victim’s body. As we will learn in the next section, the tremendous sensitivity associated with DNA technology offers investigators the opportunity to identify a perpetrator DNA types from saliva residues collected off the skin. The most efficient way to recover saliva residues from the skin is to first swab the suspect area with a rotating motion using a cotton swab moistened with distilled water. A second, dry swab is then rotated over the skin to recover the moist remains on the skin’s surface from the wet swab. The swabs are air-dried and packaged together as a single sample.

If a suspect is apprehended, the following items are routinely collected:

· 1. All clothing and any other items believed to have been worn at the time of assault.

· 2. Pubic hair combings.

· 3. Head and pubic hair standard/reference samples.

· 4. A penile swab taken within 24 hours of the assault, when appropriate to the case history.

· 5. A blood sample or buccal swab for DNA typing purposes.

The advent of DNA profiling has forced investigators to rethink what items are evidential in a sexual assault. DNA levels in the range of one-billionth of a gram are now routinely characterized in crime laboratories. In the past, scant attention was paid to the underwear recovered from a male who was suspected of being involved in a sexual assault; seminal constituents on a man’s underwear had little or no investigative value. Today, the sensitivity of DNA analysis has created new areas of investigation. It is possible to link a victim and an assailant by analyzing biological material recovered from the interior front surface of a male suspect’s underwear. This is especially important when investigations have failed to yield the presence of the suspect’s DNA on evidence recovered from the victim.

CASE FILES

A common mode of DNA transfer occurs when skin cells from the walls of a female victim’s vagina are transferred onto the suspect during intercourse. Subsequent penile contact with the inner surface of the suspect’s underwear often leads to the recovery of the female victim’s DNA from the underwear’s inner surface. The power of DNA is illustrated by a case in which the female victim of a sexual assault had consensual sexual intercourse with a male partner before being assaulted by a different male. DNA extracted from the inside front area of the suspect’s underwear revealed a female DNA profile matching that of the victim. The added bonus to investigators in this case was finding male DNA on the same underwear that matched that of the consensual partner.

Source: Based on information contained in Gary G. Verret, “Sexual Assault Cases with No Primary Transfer of Biological Material from Suspect to Victim: Evidence of Secondary and Tertiary Transfer of Biological Material from Victim to Suspect’s Undergarments,” Proceedings of the Canadian Society of Forensic Science, Toronto, Ontario, November 2001.

The persistence of seminal constituents in the vagina may help determine the time of an alleged sexual attack. Although spermatozoa in the vaginal cavity provide evidence of intercourse, important information regarding the time of sexual activity can be obtained from the knowledge that motile (living) sperm generally survive for up to six hours in the vaginal cavity of a living female. However, a successful search for motile sperm requires a microscopic examination of a vaginal smear immediately after it is taken from the victim.

A more extensive examination of vaginal collections is later made at a forensic laboratory. Nonmotile sperm may be found in a living female for up to three days after intercourse and occasionally up to six days later. Intact sperm (i.e., sperm with tails) are not normally found more than 16 hours after intercourse, but they have been found as late as 72 hours later. The likelihood of finding seminal acid phosphatase in the vaginal cavity markedly decreases with time following intercourse, with little chance of identifying this substance 48 hours after intercourse. 4 Hence, with the possibility of prolonged persistence of both spermatozoa and acid phosphatase in the vaginal cavity after intercourse, investigators should determine if and when voluntary sexual activity last occurred before the sexual assault. This information will help in evaluating the significance of finding these seminal constituents in a female victim. Blood or buccal swabs for DNA analysis should be taken from any consensual partner who had sex with the victim within 72 hours of the assault.

Another significant indicator of recent sexual activity is PSA. This semen marker normally is not detected in the vaginal cavity beyond 72 hours following intercourse. 4

Quick Review

· • A sexual assault victim should undergo a medical examination as soon as possible after the assault. At that time clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination.

· • The persistence of seminal constituents in the vagina may help determine the time of an alleged sexual attack.

Understanding DNA

The discovery of deoxyribonucleic acid (DNA), the deciphering of its structure, and the decoding of its genetic information were turning points in our understanding of the underlying concepts of inheritance. Now, with incredible speed, as molecular biologists unravel the basic structure of genes, we can create new products through genetic engineering and develop diagnostic tools and treatments for genetic disorders.

For a number of years, these developments were of seemingly peripheral interest to forensic scientists. All that changed when, in 1985, what started out as a more or less routine investigation into the structure of a human gene led to the discovery that portions of the DNA structure of certain genes are as unique to each individual as fingerprints. Alec Jeffreys and his colleagues at Leicester University, England, who were responsible for these revelations, named the process for isolating and reading these DNA markers DNA fingerprinting. As researchers uncovered new approaches and variations to the original Jeffreys technique, the terms DNA profiling and DNA typing came to be applied to describe this relatively new technology.

This discovery caught the imagination of the forensic science community because forensic scientists have long searched for ways to definitively link biological evidence such as blood, semen, hair, and tissue to a single individual. Although conventional testing procedures had gone a long way toward narrowing the source of biological materials, individualization remained an elusive goal. DNA typing has allowed forensic scientists to accomplish this goal. Although the technique is still relatively new, DNA typing has become routine in public crime laboratories. It also has been made available to interested parties through the services of a number of skilled private laboratories. In the United States, courts have overwhelmingly admitted DNA evidence and accepted the reliability of its scientific underpinnings.

GENES AND CHROMOSOMES

Hereditary material is transmitted via microscopic units called genes . The gene is the basic unit of heredity. Each gene by itself or in concert with other genes controls the development of a specific characteristic in the new individual; the genes determine the nature and growth of virtually every body structure.

gene

The basic unit of heredity, consisting of a DNA segment located on a chromosome.

The genes are positioned on chromosomes , threadlike bodies that appear in the nucleus of every body cell (see Figure 15-12 ). Almost all human cells contain forty-six chromosomes, mated in twenty-three pairs. The only exceptions are the human reproductive cells, the egg and sperm , which contain twenty-three unmated chromosomes. During fertilization, a sperm and egg combine so that each contributes twenty-three chromosomes to form the new cell ( zygote ). Hence, the new individual begins life properly, with twenty-three mated chromosome pairs. Because the genes are positioned on the chromosomes, the new individual inherits genetic material from each parent.

chromosome

A threadlike structure in the cell nucleus composed of DNA, along which the genes are located.

egg

The female reproductive cell.

sperm

The male reproductive cell.

zygote

The cell arising from the union of an egg and a sperm cell.

FIGURE 15-12 A computer-enhanced photomicrograph image of human chromosomes.

Alfred Pasieka, Science Photo Library\Photo Researchers, Inc.

Actually, two dissimilar chromosomes are involved in the determination of sex. The egg cell always contains a long chromosome known as the X chromosome ; the sperm cell may contain either a long X chromosome or a short Y chromosome . When an X-carrying sperm fertilizes an egg, the new cell has two X chromosomes (i.e., XX) and develops into a female. A Y-carrying sperm produces an XY fertilized egg and develops into a male. Because the sperm cell determines the nature of the chromosome pair, we can say that the father biologically determines the sex of the child.

X chromosome

The female sex chromosome.

Y chromosome

The male sex chromosome.

ALLELES

Just as chromosomes come together in pairs, so do the genes they bear. The position a gene occupies on a chromosome is its locus . Genes that govern a given characteristic are similarly positioned on the chromosomes inherited from the mother and father. Thus, a gene for eye color on the mother’s chromosome will be aligned with a gene for eye color on the corresponding chromosome inherited from the father. Alternative forms of genes that influence a given characteristic and are aligned with one another on a chromosome pair are known as alleles .

locus

The physical location of a gene on a chromosome.

allele

Any of several alternative forms of a gene located at the same point on a particular pair of chromosomes.

Inheritance of blood type offers a simple example of allele genes in humans. An individual’s blood type is determined by three genes, designated A, B, and O. A gene pair made up of two similar alleles—for example, AA and BB—is said to be homozygous . For example, if the chromosome inherited from the father carries the A gene and the chromosome inherited from the mother carries the same gene, the offspring will have an AA combination. Thus, when an individual inherits two similar genes from his or her parents, there is no problem in determining the blood type of that person. An individual with an AA combination will always be type A, a BB will be type B, and an OO will be type O.

homozygous

Having two identical allelic genes on two corresponding positions on a pair of chromosomes.

A gene pair made up of two different alleles—AO, for example—is said to be heterozygous . For example, if the chromosome from one parent carries the A gene and the chromosome from the other parent carries the O gene, the genetic makeup of the offspring will be AO. When two different genes are inherited, one gene will be dominant—that is, the characteristic coded for by that gene is expressed. The other gene will be recessive—that is, its characteristics remain hidden. In the case of blood types, A and B genes are dominant, and the O gene is recessive. Thus, with an AO combination, A is always dominant over O, and the individual is typed as A. Similarly, a BO combination is typed as B. In the case of AB, the genes are codominant, and the individual’s blood type will be AB. The recessive characteristics of O appear only when both recessive genes are present in combination OO, which is typed simply as O.

heterozygous

Having two different allelic genes on two corresponding positions on a pair of chromosomes.

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Quick Review

· • The gene is the basic unit of heredity. A chromosome is a threadlike structure in the cell nucleus along which the genes are located.

· • Most human cells contain forty-six chromosomes, arranged in twenty-three mated pairs. The only exceptions are the human reproductive cells, the egg and sperm, which contain twenty-three unmated chromosomes each.

· • During fertilization, a sperm and an egg combine so that each contributes twenty-three chromosomes to form the new cell, or zygote, that develops into the offspring.

· • An allele is any of several alternative forms of genes that influence a given characteristic and that are aligned with one another on a chromosome pair.

· • A heterozygous gene pair is made up of two different alleles; a homozygous gene pair is made up of two similar alleles.

· • When two different genes are inherited, the characteristic in the dominant gene’s code will be expressed. The characteristic in the recessive gene’s code will remain hidden.

WHAT IS DNA?

Inside each of 60 trillion cells in the human body are strands of genetic material called chromosomes. Arranged along the chromosomes, like beads on a thread, are nearly 25,000 genes. The gene is the fundamental unit of heredity. It instructs the body’s cells to make proteins that determine everything from hair color to susceptibility to diseases. Each gene is composed of DNA designed to carry out a single body function.

Although DNA was first discovered in 1868, scientists were slow to understand and appreciate its fundamental role in inheritance. Painstakingly, researchers developed evidence that DNA was probably the substance by which genetic instructions are passed from one generation to the next. However, the first major breakthrough in comprehending how DNA works did not occur until the early 1950s, when two researchers, James Watson and Francis Crick, deduced the structure of DNA. It turns out that DNA is an extraordinary molecule skillfully designed to control the genetic traits of all living cells, plant and animal.

STRUCTURE OF DNA

Before examining the implications of Watson and Crick’s discovery, let’s see how DNA is constructed. DNA is a polymer. A polymer is a very large molecule made by linking a series of repeating units, or monomers. In this case, the units are known as nucleotides .

nucleotide

A repeating unit of DNA consisting of one of four bases—adenine, guanine, cytosine, or thymine—attached to a phosphate-sugar group.

NUCLEOTIDES

A nucleotide is composed of a sugar molecule, a phosphorus atom surrounded by four oxygen atoms, and a nitrogen-containing molecule called a base. Figure 15-13 shows how nucleotides can be strung together to form a DNA strand. In this figure, S designates the sugar component, which is joined with a phosphate group to form the backbone of the DNA strand. Projecting from the backbone are the bases.

The key to understanding how DNA works is to appreciate the fact that only four types of bases are associated with DNA: adenine, cytosine, guanine, and thymine. To simplify our discussion of DNA, we will designate each of these bases by the first letter of their names. Hence, A will stand for adenine, C for cytosine, G for guanine, and T for thymine.

Again, notice in Figure 15-13 how the bases project from the backbone of DNA. Also, although this figure shows a DNA strand of four bases, keep in mind that in theory there is no limit to the length of the DNA strand; a DNA strand can be composed of a long chain with millions of bases. This information was well known to Watson and Crick by the time they started detailing the structure of DNA. Their efforts led them to discover that the DNA molecule is composed of two DNA strands coiled into a double helix. This can be thought of as resembling two wires twisted around each other.

As Watson and Crick manipulated scale models of DNA strands, they realized that the only way the bases on each strand could be properly aligned with each other in a double-helix configuration was to place base A opposite T and G opposite C. Watson and Crick had solved the puzzle of the double helix and presented the world with a simple but elegant picture of DNA (see Figure 15-14 ).

COMPLEMENTARY BASE PAIRING

The concept that the only arrangement possible in the double-helix configuration is the pairing of bases A to T and G to C is known as complementary base pairing. Although A–T and G–C pairs are always required, there are no restrictions on how the bases are sequenced on a DNA strand. Thus, one can observe the sequences T–A–T–T or G–T–A–A or G–T–C–A. When these sequences are joined with their complements in a double-helix configuration, they pair as follows:

FIGURE 15-13 How nucleotides can be linked to form a DNA strand. S designates the sugar component, which is joined with phosphate groups (P) to form the backbone of DNA. Projecting from the backbone are four bases: A, adenine; G, guanine; T, thymine; and C, cytosine.

FIGURE 15-14 A representation of a DNA double helix. Notice how bases G and C pair with each other, as do bases A and T. This is the only arrangement in which two DNA strands can align with each other in a double-helix configuration.

Any base can follow another on a DNA strand, which means that the number of possible sequence combinations is staggering. Consider that the average human chromosome has DNA containing 100 million base pairs. All of the human chromosomes taken together contain about three billion base pairs. From these numbers, we can begin to appreciate the diversity of DNA and, hence, the diversity of living organisms. DNA is like a book of instructions. The alphabet used to create the book is simple enough: A, T, G, and C. The order in which these letters are arranged defines the role and function of a DNA molecule.

Polymerase Chain Reaction (PCR)

Once the double-helix structure of DNA was discovered, how DNA duplicated itself prior to cell division became apparent. The concept of base pairing in DNA suggests the analogy of positive and negative photographic film. Each strand of DNA in the double helix has the same information; one can make a positive print from a negative or a negative from a positive.

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PCR PROCESS

The synthesis of new DNA from existing DNA begins with the unwinding of the DNA strands in the double helix. Each strand is then exposed to a collection of free nucleotides. Letter by letter, the double helix is re-created as the nucleotides are assembled in the proper order, as dictated by the principle of base pairing (A with T and G with C). The result is the emergence of two identical copies of DNA where before there was only one (see Figure 15-15 ). A cell can now pass on its genetic identity when it divides.

Many enzymes and proteins are involved in unwinding the DNA strands, keeping the two DNA strands apart, and assembling the new DNA strands. For example, DNA polymerases are enzymes that assemble a new DNA strand in the proper base sequence determined by the original, or parent, DNA strand. DNA polymerases also “proofread” the growing DNA double helices for mismatched base pairs, which are replaced with correct bases.

Until recently, the phenomenon of DNA replication appeared to be of only academic interest to forensic scientists interested in DNA for identification. However, this changed when researchers perfected the technology of using DNA polymerases to copy a DNA strand located outside a living cell. This laboratory technique is known as polymerase chain reaction (PCR) . Put simply, PCR is a technique designed to copy or multiply DNA strands.

polymerase chain reaction (PCR)

A technique for replicating or copying a portion of a DNA strand outside a living cell.

In PCR, small quantities of DNA or broken pieces of DNA found in crime-scene evidence can be copied with the aid of a DNA polymerase. The copying process is highly temperature dependent and can be accomplished in an automated fashion using a DNA thermal cycler (see Figure 15-16 ). Each cycle of the PCR technique results in a doubling of the DNA, as shown in Figure 15-15 . Within a few hours, thirty cycles can multiply DNA a billionfold. Once DNA copies are in hand, they can be analyzed by any of the methods of modern molecular biology. The ability to multiply small bits of DNA opens new and exciting avenues for forensic scientists to explore. It means that sample size is no longer a limitation in characterizing DNA recovered from crime-scene evidence.

FIGURE 15-15 Replication of DNA. The strands of the original DNA molecule are separated, and two new strands are assembled.

FIGURE 15-16 The DNA thermal cycler, an instrument that automates the rapid and precise temperature changes required to copy a DNA strand. Within a matter of hours, DNA can be multiplied a billionfold.

Applied Biosystems

Quick Review

· • The gene is the fundamental unit of heredity. Each gene is composed of DNA specifically designed to control the genetic traits of our cells.

· • DNA is constructed as a very large molecule made of a linked series of repeating units called nucleotides.

· • Four types of bases are associated with the DNA structure: adenine (A), guanine (G), cytosine (C), and thymine (T).

· • The bases on each strand of DNA are aligned in a double-helix configuration so that adenine pairs with thymine and guanine pairs with cytosine. This concept is known as complementary base pairing.

· • The order in which the base pairs are arranged defines the role and function of a DNA molecule.

· • DNA replication begins with the unwinding of the DNA strands in the double helix. The double helix is re-created as the nucleotides are assembled in the proper order (A with T and G with C). Two identical copies of DNA emerge from the process.

· • PCR (polymerase chain reaction) is a technique for replicating, or copying, a portion of a DNA strand outside a living cell.

CLOSER ANALYSIS POLYMERASE CHAIN REACTION

The most important feature of PCR is the knowledge that an enzyme called DNA polymerase can be directed to synthesize a specific region of DNA. In a relatively straightforward manner, PCR can be used to repeatedly duplicate or amplify a strand of DNA millions of times. As an example, let’s consider a segment of DNA that we want to duplicate by PCR:

To perform PCR on this DNA segment, short sequences of DNA on each side of the region of interest must be identified. In the example shown here, the short sequences are designated by boldface letters in the DNA segment. These short DNA segments must be available in a pure form known as a primer if the PCR technique is going to work.

The first step in PCR is to heat the DNA strands to about 94°C. At this temperature, the double-stranded DNA molecules separate completely:

The second step is to add the primers to the separated strands and allow the primers to combine, or hybridize, with the strands by lowering the test-tube temperature to about 60°C.

The third step is to add the DNA polymerase and a mixture of free nucleotides (A, C, G, T) to the separated strands. When the test tube is heated to 72°C, the polymerase enzyme directs the rebuilding of a double-stranded DNA molecule, extending the primers by adding the appropriate bases, one at a time, resulting in the production of two complete pairs of double-stranded DNA segments:

This completes the first cycle of the PCR technique, which results in a doubling of the number of DNA molecules from one to two. The cycle of heating, cooling, and strand rebuilding is then repeated, resulting in a further doubling of the DNA molecules. On completion of the second cycle, four double-stranded DNA molecules have been created from the original double-stranded DNA sample. Typically, twenty-eight to thirty-two cycles are carried out to yield more than one billion copies of the original DNA molecule. Each cycle takes less than two minutes.

DNA Typing with Short Tandem Repeats

Geneticists have discovered that portions of the DNA molecule contain sequences of letters that are repeated numerous times. In fact, more than 30 percent of the human genome is composed of repeating segments of DNA. These repeating sequences, or tandem repeats, seem to act as filler or spacers between the coding regions of DNA. Although these repeating segments do not seem to affect our outward appearance or control any other basic genetic function, they are nevertheless part of our genetic makeup, inherited from our parents. The origin and significance of these tandem repeats is a mystery, but to forensic scientists they offer a means of distinguishing one individual from another through DNA typing.

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SHORT TANDEM REPEATS (STRs)

Currently, short tandem repeat (STR) analysis has emerged as the most successful and widely used DNA-profiling procedure. STRs are locations (loci) on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule. They serve as helpful markers for identification because they are found in great abundance throughout the human genome.

short tandem repeat (STR) A region of a DNA molecule that contains short segments of three to seven repeating base pairs.

STRs normally consist of repeating sequences of three to seven bases; the entire strand of an STR is also very short, less than 450 bases long. These strands are significantly shorter than those encountered in other DNA typing procedures. This means that STRs are much less susceptible to degradation and are often recovered from bodies or stains that have been subject to extreme decomposition. Also, because of their shortness, STRs are an ideal candidate for multiplication by PCR, thus overcoming the limited-sample-size problem often associated with crime-scene evidence. Only the equivalent of eighteen DNA-containing cells is needed to obtain a DNA profile. For instance, STR profiles have been used to identify the origin of saliva residue on envelopes, stamps, soda cans, and cigarette butts.

To understand the utility of STRs in forensic science, let’s look at one commonly used STR known as TH01. This DNA segment contains the repeating sequence A–A–T–G. Seven TH01 variants have been identified in the human genome. These variants contain five to eleven repeats of A–A–T–G. Figure 15-17 illustrates two such TH01 variants, one containing six repeats and the other containing eight repeats of A–A–T–G.

During a forensic examination, TH01 is extracted from biological materials and amplified by PCR as described earlier. The ability to copy an STR means that extremely small amounts of the molecule can be detected and analyzed. Once the STRs have been copied or amplified, they are separated by electrophoresis. Here, the STRs are forced to move across a gel-coated plate under the influence of an electrical potential. Smaller DNA fragments move along the plate faster than do larger DNA fragments. By examining the distance the STR has migrated on the electrophoretic plate, one can determine the number of A–A–T–G repeats in the STR. Every person has two STR types for TH01, one inherited from each parent. Thus, for example, one may find in a semen stain TH01 with six repeats and eight repeats. This combination of TH01 is found in approximately 3.5 percent of the population. It is important to understand that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us has.

FIGURE 15-17 Variants of the short tandem repeat TH01. The upper DNA strand contains six repeats of the sequence A–A–T–G; the lower DNA strand contains eight repeats of the sequence A–A–T–G. This DNA type is known as TH01 6,8.

When examining an STR DNA pattern, one merely needs to look for a match between band sets. For example, in Figure 15-18 DNA extracted from a crime-scene stain matches the DNA recovered from one of three suspects. When comparing only one STR, a limited number of people in a population would have the same STR fragment pattern as the suspect. However, by using additional STRs, a high degree of discrimination or complete individualization can be achieved.

FIGURE 15-18 A DNA profile pattern of a suspect and its match to crime-scene DNA. From left to right, lane 1 is a DNA standard marker; lane 2 is the crime-scene DNA; and lanes 3 to 5 are control samples from suspects 1, 2, and 3, respectively. Crime-scene DNA matches suspect 2.

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MULTIPLEXING

What makes STRs so attractive to forensic scientists is that hundreds of types of STRs are found in human genes. The more STRs one can characterize, the smaller the percentage of the population from which these STRs can emanate. This gives rise to the concept of multiplexing . Using PCR technology, one can simultaneously extract and amplify a combination of different STRs.

multiplexing

A technique that simultaneously detects more than one DNA marker in a single analysis.

One STR system on the commercial market is the STR Blue Kit. This kit provides the necessary materials for amplifying and detecting three STRs (a process called triplexing): D3S1358, vWA, and FGA. The design of the system ensures that the size of the STRs does not overlap, thereby allowing each marker to be viewed clearly on an electrophoretic gel, as shown in Figure 15-19 . In the United States, the forensic science community has standardized thirteen STRs for entry into a national database known as the Combined DNA Index System (CODIS).

When an STR is selected for analysis, not only must the identity and number of core repeats be defined, but the sequence of bases flanking the repeats must also be known. This knowledge allows commercial manufacturers of STR typing kits to prepare the correct primers to delineate the STR segment to be amplified by PCR. Figure 15-20 illustrates how appropriate primers are used to define the region of DNA to be amplified. Also, a mix of different primers aimed at different STRs will be used to simultaneously amplify a multitude of STRs (i.e., to multiplex). In fact, one STR kit on the commercial market can simultaneously make copies of fifteen different STRs (see Figure 15-21 ).

DNA TYPING WITH STRs

The thirteen CODIS STRs are listed in Table 15.1 along with their probabilities of identity. The probability of identity is a measure of the likelihood that two individuals selected at random will have an identical STR type. The smaller the value of this probability, the more discriminating the STR. A high degree of discrimination and even individualization can be attained by analyzing a combination of STRs (multiplexing). Because STRs occur independent of each other, the probability of biological evidence having a particular combination of STR types is determined by the product of their frequency of occurrence in a population. This combination is referred to as the product rule (see p. 107 ). Hence, the greater the number of STRs characterized, the smaller the frequency of occurrence of the analyzed sample in the general population.

The combination of the first three STRs shown in Table 15.1 typically produces a frequency of occurrence of about 1 in 5,000. A combination of the first six STRs typically yields a frequency of occurrence in the range of 1 in 2 million for the Caucasian population, and if the top nine STRs are determined in combination, this frequency declines to about 1 in 1 billion. The combination of all thirteen STRs shown in Table 15.1 typically produces frequencies of occurrence that measure in the range of 1 in 575 trillion for Caucasian Americans and 1 in 900 trillion for African Americans. Several commercially available kits allow forensic scientists to profile STRs in the kinds of combinations cited here.

SEX IDENTIFICATION USING STRs

Manufacturers of commercial STR kits typically used by crime laboratories provide one additional piece of useful information along with STR types: the sex of the DNA contributor. The focus of attention here is the amelogenin gene located on both the X and Y chromosomes. This gene, which is actually the gene for tooth pulp, has an interesting characteristic in that it is shorter by six bases in the X chromosome than in the Y chromosome. Hence, when the amelogenin gene is amplified by PCR and separated by electrophoresis, males, who have an X and a Y chromosome, show two bands; females, who have two X chromosomes, have just one band. Typically, these results are obtained in conjunction with STR types.

FIGURE 15-19 A triplex system containing three loci: FGA, vWA, and D3S1358, indicating a match between the questioned and the standard/reference stains.

Another tool in the arsenal of the DNA analyst is the ability to type STRs located on the Y chromosome. The Y chromosome is male specific and is always paired with an X chromosome. More than twenty Y-STR markers have been identified, and a commercial kit allows for the characterization of seventeen Y chromosome STRs. When is it advantageous to seek out Y-STR types? Generally, Y-STRs are useful for analyzing blood, saliva, or a vaginal swab that is a mix originating from more than one male. For example, Y-STRs prove useful when multiple males are involved in a sexual assault.

Y-STRs

Short tandem repeats located on the human Y chromosome.

Keep in mind that STR types derived from the Y chromosome originate only from this single male chromosome. A female subject, with her XX chromosome pattern, does not contribute any DNA information. Also, unlike a conventional STR analysis that is derived from two chromosomes and typically shows two bands or peaks, a Y-STR has only one band or peak for each STR type.

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FIGURE 15-20 Appropriate primers flanking the repeat units of a DNA segment must be selected and put into place to initiate the PCR process.

FIGURE 15-21 STR profile for 15 loci.

H. Edward Grotjan, Ph.D.

For example, the traditional STR DNA pattern may be overly complex when a vaginal swab contains the semen of two males. Each STR type would be expected to show four bands, two from each male. Also complicating the appearance of the DNA profile may be the presence of DNA from skin cells from the walls of the vagina. In this circumstance, homing in on the Y chromosome greatly simplifies the appearance and interpretation of the DNA profile. Thus, when presented with a DNA mixture of two males and one female, each STR type would be expected to show six bands. However, the same mixture subjected to Y-STR analysis would show only two bands (one band for each male) for each Y-STR type.

TABLE 15.1 Thirteen CODIS STRs and Their Probability of Identities

STR	AFRICAN AMERICAN	U.S. CAUCASIAN
D3S1358	0.094	0.075
vWA	0.063	0.062
FGA	0.033	0.036
TH01	0.109	0.081
TPOX	0.090	0.195
CSF1PO	0.081	0.112
D5S818	0.112	0.158
D13S317	0.136	0.085
D7S820	0.080	0.065
D8S1179	0.082	0.067
D21S11	0.034	0.039
D18S51	0.029	0.028
D16S539	0.070	0.089

Source: The Future of Forensic DNA Testing: Predictions of the Research and Development Working Group. (Washington, DC: National Institute of Justice, Department of Justice, 2000), p. 41.

SIGNIFICANCE OF DNA TYPING

STR DNA typing has become an essential and basic investigative tool in the law enforcement community. The technology has progressed at a rapid rate and in only a few years has surmounted numerous legal challenges so that DNA typing is now vital evidence for resolving violent crimes and sex offenses. DNA evidence is impartial, implicating the guilty and exonerating the innocent.

In a number of well-publicized cases, DNA evidence has exonerated individuals who have been wrongly convicted and imprisoned. The importance of DNA analyses in criminal investigations has also placed added burdens on crime laboratories to improve their quality-assurance procedures and to ensure the correctness of their results. In fact, in several well-publicized instances, the accuracy of DNA tests conducted by government-funded laboratories has been called into question.

CLOSER ANALYSIS CAPILLARY ELECTROPHORESIS

Capillary electrophoresis has emerged as the preferred technology for characterization of STRs. Capillary electrophoresis is carried out in a thin glass column. As illustrated in the figure, each end of the column is immersed in a reservoir of buffer liquid that also holds electrodes (coated with platinum) to supply high-voltage energy. The column is coated with a gel polymer, and the DNA-containing sample solution is injected into one end of the column with a syringe. The STR fragments then move through the column under the influence of an electrical potential at a speed that is related to the length of the STR fragments. The other end of the column is connected to a detector that tracks the separated STRs as they emerge from the column. As the DNA peaks pass through the detector, they are recorded on a display known as an electropherogram.

The separation of DNA segments is carried out on the interior wall of a glass capillary tube coated with a gel polymer and kept at a constant voltage. The size of the DNA fragments determines the speed at which they move through the column. This figure illustrates the separation of three sets of STRs (called triplexing).

Quick Review

· • Short tandem repeats (STRs) are locations on the chromosome that contain short sequences that repeat themselves within the DNA molecule. They serve as useful markers for identification because they are found in great abundance throughout the human genome.

· • The entire strand of an STR is very short: less than 450 bases long. This makes STRs much less susceptible to degradation, and they are often recovered from bodies or stains that have been subjected to extreme decomposition.

· • The more STRs one can characterize, the smaller the percentage of the population from which a particular combination of STRs can emanate. This gives rise to the concept of multiplexing, in which the forensic scientist can simultaneously extract and amplify a combination of STRs.

· • With STRs, as few as eighteen DNA-containing cells are required for analysis.

Mitochondrial DNA

Typically, when one describes DNA in the context of a criminal investigation, the DNA is assumed to be the DNA in the nucleus of a cell. Actually, a human cell contains two types of DNA: nuclear and mitochondrial. The first constitutes the twenty-three pairs of chromosomes in the nuclei of our cells. Each parent contributes to the genetic makeup of these chromosomes. Mitochondrial DNA (mtDNA), on the other hand, is found outside the nucleus of the cell and is inherited solely from the mother.

Mitochondria are cell structures found in all human cells. They are the power plants of the body, providing about 90 percent of the energy that the body needs to function. A single mitochondrion contains several loops of DNA, all of which are involved in energy generation. Further, because each cell in our bodies contains hundreds to thousands of mitochondria, there are hundreds to thousands of mtDNA copies in a human cell. This compares to just one set of nuclear DNA located in that same cell.

mitochondria

Small structures outside the nucleus that supply energy to a cell.

Forensic scientists rely on mtDNA to identify a subject when nuclear DNA is significantly degraded, as in the case of charred remains, or when nuclear DNA may be present in only very small quantities (such as in a hair shaft). Interestingly, when authorities cannot obtain a reference sample from an individual who may be long deceased or missing, an mtDNA reference sample can be obtained from any maternally related relative. However, this also means that all individuals of the same maternal lineage will be indistinguishable by mtDNA analysis.

Although mtDNA analysis is significantly more sensitive than nuclear DNA profiling, forensic analysis of mtDNA is more rigorous, time consuming, and costly than nuclear DNA profiling. For this reason, only a handful of public and private forensic laboratories receive evidence for mtDNA determination. The FBI Laboratory strictly limits the types of cases in which it will apply mtDNA technology.

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One of the most publicized cases performed on human remains was the identification of the individual buried in the tomb of the Vietnam War’s unknown soldier. The remains lying in the tomb were believed to belong to 1st Lt. Michael J. Blassie, whose A-37 warplane was shot down near An Loc, South Vietnam, in 1972. In 1984, the US Army Central Identification Laboratory failed to identify the remains by physical characteristics, personal artifacts, or blood-typing results. The remains were subsequently placed in the tomb. In 1998, at the insistence of the Blassie family, the remains were disinterred for mtDNA analysis and the results were compared to references from seven families thought to be associated with the case. The remains in the tomb were subsequently analyzed and confirmed to be consistent with DNA from Lt. Blassie’s family.

CLOSER ANALYSIS FORENSIC ASPECTS OF MITOCHONDRIAL DNA

As discussed previously, nuclear DNA is composed of a continuous linear strand of nucleotides (A, C, G, and T). By contrast, mtDNA is constructed in a circular or loop configuration. Each loop contains enough A, C, G, and T (approximately 16,569 total nucleotides) to make up thirty-seven genes involved in mitochondrial energy generation.

Two regions of mtDNA have been found to be highly variable in the human population. These two regions have been designated hypervariable region I (HV1) and hypervariable region II (HV2), as shown in the figure. Again, the process for analyzing HV1 and HV2 is tedious. It involves generating many copies of these DNA hypervariable regions by PCR and then determining the order of the A-T-C-G bases constituting the hypervariable regions. This process is known as sequencing. The FBI Laboratory, the Armed Forces DNA Identification Laboratory, and other laboratories have collaborated to compile an mtDNA population database containing the base sequences from HV1 and HV2.

Once the sequences of the hypervariable regions from a case sample are obtained, most laboratories simply report the number of times these sequences appear in the mtDNA database maintained by the FBI. The mtDNA database contains about five thousand sequences. This approach permits an assessment of how common or rare an observed mtDNA sequence is in the database.

Interestingly, many of the sequences that have been determined in case work are unique to the existing database, and many types are present at frequencies of no greater than 1 percent in the database. Thus, it is often possible to demonstrate how uncommon a particular mtDNA sequence is. However, even under the best circumstances, mtDNA typing does not approach STR analysis in its discrimination power. Thus, mtDNA analysis is best reserved for samples for which nuclear DNA typing is simply not possible.

The first time mtDNA was admitted as evidence in a US court was in 1996 in the case of State of Tennessee v. Paul Ware. Here, mtDNA was used to link two hairs recovered from the crime scene to the defendant. Interestingly, in this case, blood and semen evidence were absent. Mitochondrial DNA analysis also plays a key role in the identification of human remains. An abundant amount of mtDNA is generally found in skeletal remains. Importantly, mtDNA reference samples are available from family members sharing the same mother, grandmother, great-grandmother, and so on.

Every cell in the body contains hundreds of mitochondria, which provide energy to the cell. Each mitochondrion contains numerous copies of DNA shaped in the form of a loop. Distinctive differences between individuals in their mitochondrial DNA makeup are found in two specific segments of the control region on the DNA loop, known as HV1 and HV2.

CASE FILES

In the fall of 1979, a 61-year-old patient wandered away from a US Department of Veterans Affairs medical facility. Despite an extensive search, authorities never located the missing man. More than ten years later, a dog discovered a human skull in a wooded area near the facility. DNA Analysis Unit II of the FBI Laboratory received the case in the winter of 1999. The laboratory determined that the mitochondrial DNA profile from the missing patient’s brother matched the mitochondrial DNA profile from the recovered skull and provided the information to the local medical examiner. Subsequently, the remains were declared to be those of the missing patient and returned to the family for burial.

Source: FBI Law Enforcement Bulletin 78 (2002): 21.

Quick Review

· • Mitochondrial DNA is located outside the cell’s nucleus and is inherited from the mother.

· • Mitochondria are cell structures found in all human cells. They provide most of the energy that the body needs to function.

· • Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best reserved for analyzing samples, such as hair, for which STR analysis is not possible.

Combined DNA Index System (CODIS)

Perhaps the most significant investigative tool to arise from a DNA-typing program is CODIS (Combined DNA Index System), a computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime-scene evidence, and profiles of missing people. CODIS allows crime laboratories to compare DNA types recovered from crime-scene evidence to those of convicted sex offenders and other convicted criminals.

Thousands of CODIS matches have linked serial crimes to each other and have solved crimes by allowing investigators to match crime-scene evidence to known convicted offenders. This capability is of tremendous value to investigators in cases in which the police have not been able to identify a suspect. The CODIS concept has already had a significant impact on police investigations in various states, as shown in the Case Files feature on page 401 .

Quick Review

· • CODIS is a computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime-scene evidence, and profiles of missing people.

Collection and Preservation of Biological Evidence for DNA Analysis

Since the early 1990s, the advent of DNA profiling has vaulted biological crime-scene evidence to a stature of importance that is eclipsed only by the fingerprint. In fact, the high sensitivity of DNA determinations has even changed the way police investigators define biological evidence.

Just how sensitive is STR profiling? Forensic analysts using currently accepted protocols can reach sensitivity levels as low as 125 picograms. Interestingly, a human cell has an estimated 7 picograms of DNA, which means that only eighteen DNA-bearing cells are needed to obtain an STR profile. With this technology in hand, the horizon of the criminal investigator extends beyond the traditional dried blood or semen stain to include stamps and envelopes licked with saliva, a cup or can that has touched a person’s lips, chewing gum, the sweat band of a hat, or a bedsheet containing dead skin cells. Likewise, skin cells, or epithelial cells , transferred onto the surface of a weapon, the interior of a glove, a pen, or any object recovered from a crime scene have yielded DNA results. 5 The phenomenon of transferring DNA via skin cells onto the surface of an object is called touch DNA . Again, keep in mind that, in theory, only 18 skin cells deposited on an object are required to obtain a DNA profile.

epithelial cells

The outer layer of skin cells.

touch DNA

DNA from skin cells transferred onto the surface of an object by simple contact.

Modifications to the STR technology can readily extend the level of detection down to nine or even fewer cells. A quantity of DNA that is below the normal level of detection is defined as a low copy number . However, analysts must take extraordinary care in analyzing low copy number DNA and often may find that courts will not allow this data to be admissible in a criminal trial.

low copy number

Fewer than 18 DNA-bearing cells.

COLLECTION OF BIOLOGICAL EVIDENCE

Before an investigator becomes enamored of the wonders of DNA, he or she should first realize that the crime scene must still be treated in the traditional manner. Before the collection of evidence begins, biological evidence should be photographed close up, and its location relative to the entire crime scene must be recorded through notes, sketches, and photographs. If the shape and position of bloodstains may provide information about the circumstances of the crime, an expert must immediately evaluate the blood evidence. The significance of the position and shape of bloodstains can best be ascertained when the expert has an on-site overview of the entire crime scene and can better reconstruct the movement of the individuals involved. The blood pattern should not be disturbed to collect DNA evidence before this phase of the investigation is completed.

The evidence collector must handle all body fluids and biologically stained materials with a minimum of personal contact. All body fluids must be assumed to be infectious; hence, wearing disposable latex gloves while handling the evidence is required. Latex gloves also significantly reduce the possibility that the evidence collector will contaminate the evidence. These gloves should be changed frequently during the evidence-collection phase of the investigation. Safety considerations and avoidance of contamination also call for the wearing of face masks, shoe covers, and possibly coveralls.

Blood has great evidential value when a transfer between a victim and suspect can be demonstrated. For this reason, all clothing from both victim and suspect should be collected and sent to the laboratory for examination. This procedure must be followed even when the presence of blood on a garment is not obvious to the investigator. Laboratory search procedures are far more revealing and sensitive than any that can be conducted at the crime scene. In addition, blood should also be searched for in less-than-obvious places. For example, the criminal may have wiped his or her hands on materials not readily apparent to the investigator. Investigators should look for towels, handkerchiefs, or rags that may have been used and then hidden, and should also examine floor cracks or other crevices that may have trapped blood.

CASE FILES

In 1990, a series of attacks on elderly victims was committed in Golds-boro, North Carolina, by an unknown individual dubbed the Night Stalker. During one such attack in March, an elderly woman was brutally sexually assaulted and almost murdered. Her daughter’s early arrival home saved the woman’s life. The suspect fled, leaving behind materials intended to burn the residence and the victim in an attempt to conceal the crime.

In July 1990, another elderly woman was sexually assaulted and murdered in her home. Three months later, a third elderly woman was sexually assaulted and stabbed to death. Her husband was also murdered. Although their house was set alight in an attempt to cover up the crime, fire and rescue personnel pulled the bodies from the house before it was engulfed in flames. DNA analysis of biological evidence collected from vaginal swabs from the three sexual assault victims enabled authorities to conclude that the same perpetrator had committed all three crimes. However, there was no suspect.

More than ten years after these crimes were committed, law enforcement authorities retested the biological evidence from all three cases using newer DNA technology and entered the DNA profiles into North Carolina’s DNA database. The DNA profile developed from the crime-scene evidence was compared to thousands of convicted-offender profiles already in the database.

In April 2001, a “cold hit” was made: The DNA profiles was matched to that of an individual in the convicted-offender DNA database. The perpetrator had been convicted of shooting into an occupied dwelling, an offense that requires inclusion of the convict’s DNA in the North Carolina DNA database. The suspect was brought into custody for questioning and was served with a search warrant to obtain a sample of his blood. That sample was analyzed and compared to the crime-scene evidence, confirming the DNA database match. When confronted with the DNA evidence, the suspect confessed to all three crimes.

Source: National Institute of Justice, “Using DNA to Solve Cold Cases” (NIJ Special Report), July 2002, https://www.ncjrs.gov/pdffiles1/nij/194197.pdf

PACKAGING OF BIOLOGICAL EVIDENCE

Biological evidence should not be packaged in plastic or airtight containers because accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. Each stained article should be packaged separately in a paper bag or a well-ventilated box. A red bio-hazard label must be attached to each container. If feasible, the entire stained article should be packaged and submitted for examination. If this is not possible, dried blood is best removed from a surface with a sterile cotton-tipped swab lightly moistened with distilled water from a dropper bottle.

A portion of the unstained surface material near the recovered stain must likewise be removed or swabbed and placed in a separate package. This is known as a substrate control . The forensic examiner might use the substrate swab to confirm that the results of the tests performed were brought about by the stain and not by the material on which it was deposited. However, this practice is normally not necessary when DNA determinations are carried out in the laboratory. It is critical that the collection swabs must not be packaged in a wet state. After collection, a swab must be air-dried for approximately five to ten minutes. Then it is best to place it in a swab box (see Figure 15-22 ), which has a circular hole to allow air circulation. The swab box can then be placed in a paper or manila envelope.

substrate control

An unstained object adjacent to an area on which biological material has been deposited.

FIGURE 15-22 Air-dried swabs are placed in a swab box for delivery to the forensic laboratory.

Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com

TABLE 15.2 Location and Sources of DNA at Crime Scenes

EVIDENCE	POSSIBLE LOCATION OF DNA ON THE EVIDENCE	SOURCE OF DNA
Baseball bat or similar weapon	Handle, end	Sweat, skin, blood, tissue
Hat, bandanna, or mask	Inside	Sweat, hair, dandruff
Eyeglasses	Nose or ear pieces, lens	Sweat, skin
Facial tissue, cotton swab	Surface area	Mucus, blood, sweat, semen, ear wax
Dirty laundry	Surface area	Blood, sweat, semen
Toothpick	Tips	Saliva
Used cigarette	Cigarette butt	Saliva
Stamp or envelope	Licked area	Saliva
Tape or ligature	Inside/outside surface	Skin, sweat
Bottle, can, or glass	Sides, mouthpiece	Saliva, sweat
Used condom	Inside/outside surface	Semen, vaginal and/or rectal cells
Blanket, pillow, sheet	Surface area	Sweat, hair, semen, urine, saliva
“Through and through” bullet	Outside surface	Blood, tissue
Bite mark	Person’s skin or clothing	Saliva
Fingernail, partial fingernail	Scrapings	Blood, sweat, tissue

Source: National Institute of Justice, US Department of Justice.

All packages containing biological evidence should be refrigerated or stored in a cool location out of direct sunlight until delivery to the laboratory. However, one common exception is blood mixed with soil. Microbes present in soil rapidly degrade DNA. Therefore, blood in soil must be stored in a clean glass or plastic container and immediately frozen.

OBTAINING DNA REFERENCE SPECIMENS

Biological evidence attains its full forensic value only when an analyst can compare each of its DNA types to known DNA samples collected from victims and suspects. For this purpose, at least 7 cc of whole blood should be drawn from individuals by a qualified medical professional. The blood sample should be collected in a sterile vacuum tube containing the preservative EDTA (ethylenediamine tetraacetic acid). In addition to serving as a preservative, EDTA inhibits the activity of enzymes that degrade DNA. The tubes must be kept refrigerated (not frozen) while awaiting transportation to the laboratory. In addition to extracting blood, there are other ways of obtaining standard/reference DNA specimens. The least intrusive method for obtaining a DNA standard/reference, one that nonmedical personnel can readily use, is the buccal swab. Cotton swabs are inserted into the subject’s mouth, and the inside of the cheek is vigorously swabbed, resulting in the transfer of buccal cells onto the swab.

buccal cells

Cells from the inner cheek lining.

With the increasing need for collection and analysis of DNA samples in forensic investigations, collection and long-term storage of DNA has become an important consideration. FTA brand paper is a type of commercially available filter paper loaded with a mix of reagents on which DNA samples can be stored. An FTA paper card has been impregnated with a chemical that protects DNA from bacterial enzyme breakdown. The fibers of the paper can entrap the DNA for at least ten years without refrigeration, allowing it to be easily stored. Figure 15-23 illustrates the collection of a buccal swab and its transfer onto an FTA card for storage.

If an individual is not available to give a DNA standard/reference sample, some interesting alternative sources are available, including the individual’s toothbrush, comb or hairbrush, razor, soiled laundry, used cigarette butts, and earplugs. Any of these items may contain a sufficient quantity of DNA for typing. Interestingly, as investigators worked to identify the remains of victims of the World Trade Center attack on September 11, 2001, the families of the missing were asked to supply the New York City DNA Laboratory with these types of items in an effort to match recovered DNA with human remains.

CONTAMINATION OF DNA EVIDENCE

One key concern while collecting a DNA-containing specimen is contamination. Contamination can occur by introducing foreign DNA onto a stain through coughing or sneezing during the collection process, or there can be a transfer of DNA when items of evidence are incorrectly placed in contact with each other during packaging. Fortunately, an examination of DNA band patterns in the laboratory readily reveals the presence of contamination. For example, with an STR, one will expect to see a two-band pattern. More than two bands suggests a mixture of DNA from more than one source.

Crime-scene investigators can take some relatively simple steps to minimize the contamination of biological evidence:

· 1. Change gloves before handling each new piece of evidence.

· 2. Collect a substrate control for possible subsequent laboratory examination.

· 3. Pick up small items of evidence such as cigarette butts and stamps with clean forceps. Use disposable forceps so that they can be discarded after a single evidence collection.

· 4. Always package each item of evidence in its own well-ventilated container.

A common occurrence at crime scenes is to suspect the presence of blood but not be able to observe any with the naked eye. In these situations, the common test of choice is luminol or Bluestar. Interestingly, luminol and Bluestar do not inhibit the ability to detect and characterize STRs. 2 Therefore, they can be used to locate traces of blood and areas that have been washed nearly free of blood without compromising the potential for DNA typing.

Quick Review

· • Packaging of bloodstained evidence in plastic or airtight containers must be avoided because the accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. Each stained article should be packaged separately in a paper bag or in a well-ventilated box.

· • The least intrusive method for obtaining a DNA standard/reference is the buccal swab. In this procedure, cotton swabs are inserted into the subject’s mouth, and the inside of the cheek is vigorously swabbed, resulting in the transfer of cells from the inner cheek lining onto the swab.

WebExtra 15.15

DNA Forensics www.mycrimekit.com

WebExtra 15.16

Step into the Role of the First Responding Officer at a Sexual Assault Scene www.mycrimekit.com

WebExtra 15.17

Assume the Duties of an Evidence Collection Technician at a Sexual Assault Scene www.mycrimekit.com

VIRTUAL LAB DNA Analysis

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VIRTUAL LAB DNA Analysis

To perform a virtual bloodstain analysis, go to www.pearsoncustom.com/us/vlm/

FIGURE 15-23 (a) A buccal swab is collected by rubbing each cheek for 15 seconds. (b) A protective film is peeled off the FTA card. (c) The swab is snapped in place against the FTA paper. (d) The FTA card is removed from the collection device and stored.

Courtesy GE Healthcare Bio-Sciences Corp. (GEHC), Piscataway, NJ, www.whatman.com

CASE FILES

A woman alleged that she had been held in an apartment against her will and sexually assaulted by a male friend. During the course of the assault, a contact lens was knocked from the victim’s eye. After the assault, she escaped, but out of fear from threats made by her attacker, she did not report the assault to the police for three days. When the police examined the apartment, they noted that it had been thoroughly cleaned. A vacuum cleaner bag was seized for examination, and several pieces of material resembling fragments of a contact lens were discovered within the bag.

In the laboratory, approximately 20 nanograms of human DNA were recovered from the contact lens fragments. Because cells from both a person’s eyeballs and the interior of the eyelids are naturally replaced every 6 to 24 hours, both were potential sources for the DNA found. The DNA profile originating from the fragments matched the victim, thus corroborating the victim’s account of the crime. The estimated frequency of occurrence in the population for the nine matching STRs is approximately 1 in 850 million. The suspect subsequently pleaded guilty to the offense. *

STR Locus	Victim’s DNA Type	Contact Lens
D3S1358	15,18	15,18
FGA	24,25	24,25
vWA	17,17	17,17
THO1	6,7	6,7
F13A1	5,6	5,6
fes/fps	11,12	11,12
D5S818	11,12	11,12
D13S317	11,12	11,12
D7S820	10,12	10,12

* Based on information contained in R. A. Wickenheiser and R. M. Jobin, “Comparison of DNA Recovered from a Contact Lens Using PCR DNA Typing.” Canadian Society of Forensic Science Journal 32 (1999): 67.