The Green River Killer

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The Green River Killer

This case takes its name from the Green River, which flows through Washington state and empties into Puget Sound in Seattle. In 1982, within six months the bodies of five females were discovered in or near the river. Most of the victims were known prostitutes who were strangled and apparently raped. As police focused their attention on an area known as Sea-Tac Strip, a haven for prostitutes, girls mysteriously disappeared with increasing frequency. By the end of 1986, the body count in the Seattle region rose to 40, all of whom were believed to have been murdered by the Green River Killer. As the investigation pressed on into 1987, the police renewed their interest in one suspect, Gary Ridgway, a local truck painter. Ridgway had been known to frequent the Sea-Tac Strip. Interestingly, in 1984 Ridgway had actually passed a lie detector test. Now with a search warrant in hand, police searched the Ridgway residence and also obtained hair and saliva samples from Ridgway. Again, insufficient evidence caused Ridgway to be released

from custody. With the exception of one killing in 1998, the murder spree stopped in 1990, and the case remained

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dormant for nearly ten years. But the advent of DNA testing brought renewed vigor to the investigation. In 2001, semen samples collected from three early victims of the

Green River Killer were compared to Ridgway’s saliva that had been collected in 1987. The DNA profiles matched and the police had their man. An added forensic link to Ridgway was made by the location of minute amounts of spray paint on the clothing of six victims that compared to paints collected from Ridgway’s workplace. Ridgway avoided the death penalty by confessing to the murders of 48 women.

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

R O D D Y , A N T H O N Y I S A A C 3 7 2 7 B U

 

 

After studying this chapter you should be able to: • List the A-B-O antigens and antibodies found in the blood for

each of the four blood types: A, B, AB, and O

• Understand and describe how whole blood is typed

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

• Understand the concept of antigen–antibody interactions and how it is applied to species identification and drug identification

• Explain the differences between monoclonal and polyclonal antibodies

• Contrast chromosomes and genes

• Learn how the Punnett square is used to determine the genotypes and phenotypes of offspring

• List the laboratory tests necessary to characterize seminal stains

• Explain how suspect blood and semen stains are to be properly preserved for laboratory examination

• Describe the proper collection of physical evidence in a rape investigation

forensic serology

acid phosphatase agglutination allele antibody antigen antiserum aspermia chromosome deoxyribonucleic

acid (DNA) egg erythrocyte gene genotype hemoglobin heterozygous homozygous hybridoma cells locus luminol monoclonal antibodies oligospermia phenotype plasma polyclonal antibodies precipitin serology serum sperm X chromosome Y chromosome zygote

KEY TERMS

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

R O D D Y , A N T H O N Y I S A A C 3 7 2 7 B U

 

 

plasma The fluid portion of unclotted blood

DNA Abbreviation for deoxyribonucleic acid—the molecules carrying the body’s genetic information; DNA is double stranded in the shape of a double helix

242 CHAPTER 10

In 1901, Karl Landsteiner announced one of the most significant discoveries of the 20th century— the typing of blood—a finding that 29 years later earned him a Nobel Prize. For years physicians had attempted to transfuse blood from one individual to another. Their efforts often ended in failure be- cause the transfused blood tended to coagulate 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 had the key for properly matching the blood of a donor to a recipient. One blood type cannot be mixed with a different blood type without disas- trous consequences. This discovery, of course, had important implications for blood transfusion and the millions of lives it has since saved. Meanwhile, Landsteiner’s findings had opened up a com- pletely 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 was demonstrated, and shortly thereafter, numerous blood factors or groups were discovered. More than a hundred different blood factors have been shown to exist. 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 offer- ing the best means for linking blood to an individual. What made these factors so attractive to the forensic scientist was that in theory no two individuals, except for identical twins, could be ex- pected 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 high frequency of occurrence of bloodstains at crime scenes, especially crimes of the most serious nature—that is, homicides, assaults, and rapes. Consider, for example, a transfer of blood between the victim and assailant during a strug- gle; that is, the victim’s blood is transferred to the suspect’s garment, or vice versa. If the crimi- nalist could individualize human blood by identifying all of its known factors, the result would be evidence of the strongest kind for linking the suspect to the crime scene.

The advent of DNA technology has dramatically altered the approach of forensic scientists to- ward 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). The individualization of dried blood and other biological evidence, now a reality, has significantly altered the role that crime lab- oratories play in criminal investigations. As we will learn in the next chapter, the high sensitivity of DNA analysis has even altered the type of materials collected from crime scenes in the search for DNA. The next chapter is devoted to discussing recent breakthroughs in associating blood and semen stains with a single individual through characterization of DNA. This chapter focuses on underlying biological concepts that forensic scientists historically relied on as they sought to char- acterize and individualize biological evidence before the dawning of the age of DNA.

The Nature of Blood The word blood actually refers to a highly complex mixture of cells, enzymes, proteins, and in- organic substances. The fluid portion of blood is called plasma. Plasma is composed principally of water and accounts for 55 percent of blood content. Suspended in the plasma are solid materi- als consisting chiefly of cells—that is, red blood cells (erythrocytes), white blood cells (leuko- cytes), 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 one were to remove the clotted material, a pale yellowish liquid known as serum would be left.

Obviously, considering the complexity of blood, any discussion of its function and chemistry would have to be extensive, extending beyond the scope of this text. It is certainly far more rele- vant at this point to concentrate our discussion on the blood components that are directly perti- nent to the forensic aspects of blood identification—the red blood cells and the blood serum.

Antigens and Antibodies Functionally, red blood cells transport oxygen from the lungs to the body tissues and in turn re- move carbon dioxide from tissues by transporting it back to the lungs, where it is exhaled. How- ever, for reasons unrelated to the red blood cell’s transporting mission, on the surface of each cell

erythrocyte A red blood cell

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

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

R O D D Y , A N T H O N Y I S A A C 3 7 2 7 B U

 

 

serology The study of antigen–antibody reactions

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

antiserum Blood serum that contains specific antibodies

antibody A protein that destroys or inactivates a specific antigen; antibodies are found in the blood serum

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

FORENSIC SEROLOGY 243

are millions of characteristic chemical structures called antigens. Antigens impart blood-type characteristics to the red blood cells. Blood antigens are grouped into systems depending on their relationship to one another. More than 15 blood antigen systems have been identified to date; of these, the A-B-O and Rh systems are the most important.

If an individual is type A, this simply indicates 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 con- tain both A and B antigens. Type O individuals have neither A nor B antigens on their cells. Hence, the presence or absence of the 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 designated as the Rh factor, or D antigen. People with 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 determined in testing for the compatibility of the donor and recipient.

Serum is important because it contains certain proteins known as antibodies. The funda- mental 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 A antigen, anti-B for B antigen, and anti-D for D antigen. The serum-containing antibody is referred to as the antiserum, meaning a serum that reacts against something (antigens).

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 immediately 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 as clumping or agglutination (see Figure 10–1).

Let’s look a little more closely at this phenomenon. In normal blood, shown in Figure 10–2(a), antigens on red blood cells and antibodies coexist without destroying each other because the anti- bodies present are not specific toward any of the antigens. However, suppose a foreign serum added to the blood introduces a new antibody. The occurrence of a specific antigen–antibody reaction im- mediately causes the red blood cells to link together, or agglutinate, as shown in Figure 10–2(b).

Evidently, nature has taken this situation into account because when we examine the serum of type A blood, we find anti-B and 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, transfusing 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.

Blood Typing The term serology is used to describe a broad scope of laboratory tests that use specific antigen and serum antibody reactions. The most widespread application of serology is the typing of whole blood for its A-B-O identity. In determining the A-B-O blood type, only two antiserums are needed—anti-A and anti-B. For routine blood typing, both of these antiserums are commercially available.

Table 10–1 summarizes how the identity of each of the four blood groups is established when the blood is tested with anti-A and anti-B serum. Type A blood is agglutinated by anti-A serum;

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

R O D D Y , A N T H O N Y I S A A C 3 7 2 7 B U

 

 

244 CHAPTER 10

FIGURE 10–2 (a) Microscopic view of normal red blood cells (500�). (b) Microscopic view of agglutinated red blood cells (500�). Courtesy J. C. Revy, Phototake NYC

A

B

A A

Anti-B

Red blood cells containing A antigens do not combine with B antibodies

B B

B

B

B

Anti-B

Red blood cells containing B antigens are agglutinated or clumped together in the presence of B antibodies

FIGURE 10–1

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

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FORENSIC SEROLOGY 245

TABLE 10–1 Identification of Blood with Known Antiserum

Anti-A Serum �

Whole Blood

Anti-B Serum �

Whole Blood Antigen Present Blood Type

� � A A � � B B � � A and B AB � � Neither A nor B O

Note: � shows agglutination; – shows absence of agglutination.

TABLE 10–2 Identification of Blood with Known Cells

A Cells � Blood B Cells � Blood Antibody Present Blood Type

� � Anti-A B � � Anti-B A � � Both anti-A and anti-B O � � Neither anti-A nor anti-B AB

Note: � shows agglutination; – shows absence of agglutination.

type B blood is agglutinated by anti-B serum; type AB blood is agglutinated by both anti-A and anti-B; and type O blood is not agglutinated by either the anti-A or anti-B serum.

The identification of natural antibodies present in blood offers another route to the determi- nation of blood type. Testing blood for the presence of anti-A and anti-B requires using red blood cells that have known antigens. Again, these cells are commercially available. Hence, when A cells are added to a blood specimen, agglutination occurs only in the presence of anti-A. Simi- larly, B cells agglutinate only in the presence of anti-B. All four A-B-O types can be identified in this manner by testing blood with known A and B cells, as summarized in Table 10–2.

The population distribution of blood types varies with location and race throughout the world. In the United States, a typical distribution is as follows:

O A B AB

43% 42% 12% 3%

Immunoassay Techniques The concept of a specific antigen–antibody reaction is finding application in other areas unrelated to the blood typing of individuals. Most significantly, this approach has been extended to the detection of drugs in blood and urine. Antibodies that react with drugs do not naturally exist; how- ever, 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 stim- ulating the animal to produce antibodies (see Figure 10–3). The recovered blood serum of the animal will contain antibodies that are specific or nearly specific to the drug.

HO

HO

NCH3 O

HO

HO

NCH3 O

Drug Protein carrier Drug antibodies

FIGURE 10–3

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

R O D D Y , A N T H O N Y I S A A C 3 7 2 7 B U

 

 

246 CHAPTER 10

Several immunological assay techniques are commercially available for detecting drugs through an antigen–antibody reaction. One such technique, the enzyme-multiplied immunoassay technique (EMIT), has gained widespread popularity among toxicologists because of its speed and high sensitivity for detecting drugs in urine.

Enzyme-Multiplied Immunoassay Technique (EMIT) A typical EMIT analysis begins by adding to a subject’s urine antibodies that bind to a particular type or class of drug being looked for. This is followed by adding to the urine a chemically labeled version of the drug. As shown in Figure 10–4, a competition will ensue between the labeled and unlabeled drug (if it’s present in the subject’s urine) to bind with the antibody. If this competition does occur in a person’s urine, it signifies that the urine screen test was positive for the drug be- ing tested. For example, to check someone’s urine for methadone, the analyst would add methadone antibodies and chemically labeled methadone to the urine. Any methadone present in the urine immediately competes with the labeled methadone to bind with the methadone anti- bodies. The quantity of chemically labeled methadone left uncombined is then measured, and this value is related to the concentration of methadone originally present in the urine.

One of the most frequent uses of EMIT in forensic laboratories has been for screening the urine of suspected marijuana users. The primary pharmacologically active agent in marijuana is tetrahydrocannabinol, or THC. To facilitate the elimination of THC, the body converts it to a se- ries of substances called metabolites that are more readily excreted. The major THC metabolite found in urine is a substance called THC-9-carboxylic acid. Antibodies against this metabolite are prepared for EMIT testing. Normally the urine of marijuana users contains a very small quantity of THC-9-carboxylic acid (less than one-millionth of a gram); however, this level is readily detected by EMIT.

The greatest problem with detecting marijuana in urine is interpretation. Although smoking marijuana will result in the detection of THC metabolite, it is difficult to determine when the in- dividual actually used marijuana. In individuals who smoke marijuana frequently, detection is possible within two to five days after the last use of the drug. However, some individuals may yield positive results up to thirty days after the last use of marijuana.

Currently, thousands of individuals regularly submit to urinalysis tests for the presence of drugs of abuse. These individuals include military personnel, transportation industry employees, police and corrections personnel, and subjects requiring preemployment drug screening. Immunoassay testing for drugs has proven quite suitable for handling the large volume of

*

1 Labeled drug

Antibody

Antibody

Drug present

in urine

No drug

present in urine

* * * *

*

*

*

*

* * *

*

*

*

*

FIGURE 10–4 In the EMIT assay, a drug that may be present in a urine specimen will compete with added labeled drugs for a limited number of antibody binding sites. The labeled drugs are indicated by an asterisk. Once the competition for antibody sites is completed, the number of remaining unbound labeled drug is proportional to the drug’s concentration in urine.

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

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monoclonal antibodies A collection of identical antibodies that interact with a single antigen site

polyclonal antibodies Antibodies produced by injecting animals with a specific antigen; a series of antibodies are produced responding to a variety of different sites on the antigen

FORENSIC SEROLOGY 247

specimens that must be rapidly analyzed for drug content on a daily basis. Testing laboratories have access to many commercially prepared sera arising from animals being injected with any one of a variety of drugs. A particular serum that has been added to a urine specimen is designed to interact with opiates, cannabinoids, cocaine, amphetamines, phencyclidine, barbiturates, methadone, or other drugs. A word of caution: immunoassay is only presumptive in nature, and its result must be confirmed by additional testing. Specifically, the confirmation test of choice is gas chromatography-mass spectrometry, which is described in more detail in Chapter 9.

Monoclonal Antibodies As we have seen in the previous section, when an animal such as a rabbit or mouse is injected with an antigen, the animal responds by producing antibodies designed to bind to the invading antigen. However, the process of producing antibodies designed to respond to foreign antigens is complex. For one, an antigen typically has structurally different sites to which an antibody may bind. So when the animal is actively producing attack antibodies, it produces a series of different antibodies, all of which are designed to attack some particular site on the antigen of interest. These antibodies are known as polyclonal antibodies. However, the disadvantage of polyclonal antibodies is that an animal can produce antibodies that vary in composition over time. As a result, different batches of polyclonals may vary in their specificity and their ability to bind to a particular antigen site.

As the technologies associated with forensic science have grown in importance, a need has developed, in some instances, to have access to antibodies that are more uniform in their compo- sition and attack power than the traditional polyclonals. This is best accomplished by adopting a process in which an animal will produce antibodies designed to attack one and only one site on an antigen. Such antibodies are known as monoclonal antibodies. How can such monoclonals be produced? The process begins by injecting a mouse with the antigen of interest. In response, the mouse’s spleen cells will produce antibodies to fight off the invading antigen. The spleen cells are removed from the animal and are fused to fast-growing blood cancer cells to produce hybridoma cells. The hybridoma cells are then allowed to multiply and are screened for their spe- cific antibody activity. The hybridoma cells that bear the antibody activity of interest are then selected and cultured. The rapidly multiplying cancer cells linked to the selected antibody cells produce identical monoclonal antibodies in a limitless supply (see Figure 10–5).

Monoclonal antibodies are being incorporated into commercial forensic test kits with in- creasing frequency. Many immunoassay test kits for drugs of abuse are being formulated with monoclonal antibodies. Also, a recently introduced test for seminal material that incorporates a monoclonal antibody has found wide popularity in crime laboratories (see page 236).

As a side note, in 1999 the U.S. Food and Drug Administration approved a monoclonal drug treatment for cancer. Rituxin is a nontoxic monoclonal antibody designed to attack and destroy cancerous white blood cells containing an antigen designated as CD20. Other monoclonal drug treatments are in the pipeline. Monoclonals are finally beginning to fulfill their long-held expec- tation as medicine’s version of the “magic bullet.”

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 of human origin, how closely can it be associated with a particular individual?

Color Tests The determination of blood is best made by means of a preliminary color test. For many years, the most commonly used test for this purpose 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 in its place (this test is also known as the Kastle-Meyer color test).1 Both the benzidine and Kastle-Meyer color tests are based on the

1 S. Tobe et al., “Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA,” Journal of Forensic Sciences 52 (2007): 102–109.

hybridoma cells Fused spleen and tumor cells; used to produce identical monoclonal antibodies in a limitless supply

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, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.

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WEBEXTRA 10.1 See a Color Test for Blood www.mycrimekit.com

hemoglobin A red blood cell protein that transports oxygen in the bloodstream; it is responsible for the red color of blood

248 CHAPTER 10

Antigen

Antibodies Spleen cells

Malignant blood cells

Spleen cell

Hybridoma cells

Monoclonal antibodies

1. Inject mouse or rabbit with antigen.

2. Remove spleen and isolate spleen cells, which produce antibodies to the antigen of interest.

3. Fuse spleen cells with malignant cells, which grow well in culture.

4. Grow hybrid cells and isolate ones that produce the antibody of interest.

5. Culture the hybrid cells to create a virtually limitless supply of antibodies.

FIGURE 10–5 Steps required to produce monoclonal antibodies.

observation that blood hemoglobin possesses peroxidase-like activity. Peroxidases are enzymes that accelerate the oxidation of several classes of organic compounds by peroxides. When a bloodstain, phenolphthalein reagent, and hydrogen peroxide are mixed together, the blood’s hemoglobin causes the formation of 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, it is unlikely that such materials will 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 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 is indicative of blood.

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precipitin An antibody that reacts with its corresponding antigen to form a precipitate

luminol The most sensitive chemical test that is capable of presumptively detecting bloodstains diluted to as little as 1 in 100,000; its reaction with blood emits light and thus requires the result to be observed in a darkened area

FORENSIC SEROLOGY 249

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