Understanding blood

Blood is a complex substance. The liquid portion of whole blood, called plasma, contains proteins, enzymes, clotting factors, electrolytes, and various types of cells. Blood cells come in three basic types:

• Leucocytes, or white blood cells (WBCs)
• Erythrocytes, or red blood cells (RBCs)
• Platelets, or tiny cells involved in blood clotting

Whenever whole blood is permitted to clot, and the clotted material is removed, the remaining yellowish liquid is called serum. Serum contains most of the proteins and enzymes of plasma, but none of the cells or clotting factors that were consumed during the clotting process.

Blood typing: The ABO system

From a forensic serologist’s point of view, the two most important components of blood are the RBCs and the serum. From these, serologists can determine the blood type of any blood samples or bloodstains.

The RBCs contain extremely important molecules called antigens, which not only instigate immune reactions within the body but also determine blood type. Antigens are designated as either A or B. People with Type A blood have A antigens on their RBCs, and those with Type B blood have B antigens. People with Type AB have A and B antigens, and RBCs of people with Type O blood have neither antigen.

Another antigen in the blood is called the Rhesus or Rh factor, which sometimes is referred to as the D antigen. If your RBCs have these antigens, your blood is deemed Rh positive, and if they don’t, Rh negative. So those with A-positive blood possess the A antigen and the Rh (D) antigen on their RBCs. People who have O-negative blood have neither the A or B, nor the Rh antigens on their RBCs.

Another important factor to keep in mind is that blood serum contains specialized proteins called antibodies. The key point in understanding blood typing is that for every antigen there is a corresponding antibody. An antibody is highly specific, meaning that it recognizes and reacts with only its specific antigen. When an antibody meets its corresponding antigen, the two combine to form an antigen-antibody complex. This reaction is the basis for the blood-typing procedure.

Serologists test each crime-scene blood sample separately and determine its type. At crime scenes where more than one person sheds blood, such blood-typing helps investigators reconstruct the crime scene. Blood typing was critical to crime-scene reconstruction in the Jeffrey MacDonald case (see Chapter 20).

Answering the first question: Is it blood?

To determine whether a given sample actually is blood (or some other substance), the serologist conducts tests of two basic types: presumptive and confirmatory. Presumptive tests typically are cheaper and faster. When they are positive, presumptive tests indicate a likelihood that blood is present but don’t establish that as fact. Confirmatory testing then is needed to be certain. When presumptive tests are negative, blood is not present, and the more expensive and time-consuming confirmatory tests can be avoided.

Knowing when blood’s really human

When blood is found at a crime scene, the ME then must determine whether the blood is indeed from a human and not from a dog or cat — or a duck-billed platypus, for that matter. Only after this determination is made can further testing be carried out to discover whose blood it is.

Tests used for finding out which species blood came from are antigen-antibody reactions, like the ones used for blood typing (see the earlier section “Blood typing: The ABO system”). The difference, however, is that an antiserum (a substance that contains antibodies against a specific antigen) must be created that reacts with antigens specific to humans rather than with the A and B RBC antigens. That means a specific antibody to a specific human antigen is created so that the resulting reaction, or lack of one, will determine whether the blood is human.

A human antiserum is prepared by injecting human antigen (human blood) into a rabbit or other animal and then allowing enough time for the animal to produce sufficient antibodies against the antigen. The animal’s blood, now rich in antihuman antibodies, is removed, and that antiserum is isolated for use in testing blood samples to determine whether they are from humans.

If a solution that contains an antiserum is brought into contact with a blood sample that contains the antigen it was designed to react against, a reaction occurs. This reaction produces an antigen-antibody complex that precipitates or falls out of solution and results in a visible line of precipitation settling out where the two solutions come into contact.

In other words, if an antiserum to human blood comes into contact with a solution that contains human blood, the reaction forms a visible line of precipitant or solid material between the two solutions. If, on the other hand, the blood is not human, no reaction occurs, and no line is created.

The modern crime lab contains antiserums to a variety of common animal bloods. Dog, cat, deer, cow, and sheep antiserums usually are readily available. Using these antiserums, a serologist can determine the species that shed the blood, a factor that can be important evidence in and of itself.

Narrowing the focus: Whose blood is it?

After determining that a blood sample is human, the serologist sets about determining its type. Standard blood typing uses liquid blood, and a positive reaction is indicated by agglutination, or clumping, of the RBCs. Agglutination can occur only if the blood is liquid and if the RBCs are intact.

However, crime-scene blood rarely is liquid; it’s more likely to be a stain — that is, clotted and dried — and the RBCs therefore have fragmented. Because the RBCs are not intact, they can’t agglutinate, and thus no antigen-antiserum typing reaction can be verified. The serologist cleverly gets around this problem with a technique that draws out the remaining antigens.

Absorption-elution is the process that extracts the antigens in these four steps:

1. The bloodstained material is treated with blood antiserums.

   The antibodies in the antiserums combine with the antigens.

2. The material is washed.

   This step removes any excess antiserum-containing antibodies.

3. The sample undergoes elution.

   Elution is a process that breaks down antigen-antibody bonds by heating them, thus freeing the antigen and the antibodies from one another. The antibodies that were bound to the antigens are washed off.

4. The eluted antibodies then are tested against known blood antigens, and their reactions are observed.

   The antigens the antibodies react with reveal which antigens are present in the original unknown sample.

A serologist confronted with a bloodstained shirt determines that the blood is human and goes through the steps in the previous list. If the stain is from someone with Type A blood, when the anti-A and anti-B serums are added in the first step, the blood from the stain reacts only with the anti-A antibodies because it has only A antigens. After washing (Step 2), only the complex of A antigens and the anti-A antibodies remains. Elution (Step 3) further separates the antigens and antibodies, thus freeing the anti-A antibodies from the stain. Testing the antibodies against blood samples from known blood types then reveals to the serologist a reaction with only Type A blood, meaning that the original sample must have been Type A.

By simply typing the blood at a crime scene, investigators narrow their suspect list and completely exonerate some suspects by using the population distribution information for the four ABO blood types in Table 14-1.

Besides determining the ABO type, serologists are able to further individualize blood samples. RBCs contain more proteins, enzymes, and antigens than those used in the ABO classification system. These include antigens with such catchy names as Duffy, Kell, and Kidd and intracellular enzymes such as adenylate kinase, erythrocyte acid phosphatase, and the very useful phosphoglucomutase (PGM).

PGM is an enzyme that appears in many different forms, or isoenzymes, and at least ten of them are fairly common. Regardless of ABO type, a particular individual can have any combination of the isoenzymes of PGM. The ME and the serologist use that fact to further narrow the list of suspects for further DNA analyses and confirmation that they were capable of leaving a particular bloodstain.

For example, say that a stain is Type AB and has PGM 2. The ME knows the AB blood type is found in only 3 percent (see Table 14-1) of the population, and PGM 2 is found in only 6 percent of people. Because these two factors are inherited independently, the probability of a particular individual being Type AB, PGM 2 is only 0.18 percent or less than 2 per 1,000.

If the police find blood at the scene that matches the blood of a suspect who has Type AB, PGM 2 blood, the probability that that suspect is not the perpetrator is 2 in 1,000. Although not perfect, those odds still are much better than a coin toss. DNA testing, which I discuss in Chapter 15, then is used to further individualize the sample.

Inheriting your blood type

ABO blood types, or phenotypes, come in only four varieties: A, B, AB, and O. But, for some blood types two genotypes, or gene pairings, are possible. A phenotype is what something looks like (in this case the ABO blood type), while the genotype is the underlying genetic pattern. We receive our ABO genes from our parents, one from Dad and one from Mom.

The important thing to know in this system is that A and B genes are codominant (equally dominant), while the O gene is recessive. So someone who receives an A gene from one parent and an O gene from the other has Type A blood, but not Type O, because the A gene is dominant (see Table 14-2).

People with Type O blood must have an OO genotype. They can have neither an A nor a B gene because having one or the other dominates the O gene and produces either Type A or Type B blood.

A person with Type A blood can either receive an A gene from each parent and thus have an AA genotype or an A gene from one parent and an O gene from the other for an AO genotype. Remember, A is dominant, so when it is paired with the recessive O gene, the A gene determines blood type. People with the AA and AO genotypes both have Type A blood, but genetically speaking, they’re different.

Type A parents who have AA genotypes can provide only A genes to their offspring, because all their eggs or sperm have an A gene. But Type A parents who have AO genotypes can provide either an A gene or an O gene to their offspring, because half their eggs or sperm have an A gene, and the other half have an O gene. When both parents are Type A, several possibilities exist for the genotype their offspring will have. Check out the possibilities in the Punnet Squares in Figure 14-1.

In each of the scenarios presented in Figure 14-1, the child’s blood type is Type A, except when both parents donate an O gene. In the latter case, the child’s genotype and blood type (phenotype) respectively are OO and Type O. These parents can’t have any offspring who have Type B phenotype or BB, BO, or AB genotypes, because neither parent has a B gene to donate.

Determining fatherhood

Blood typing can exclude paternity but cannot absolutely verify it. For example, a man with Type AB blood can’t father a child with Type O blood. So if a child has Type O blood, all men with the Type AB are ruled out as the child’s father. A man with Type A (genotypes AA or AO) blood can be the father, but only if he has an AO genotype. Men who have AA genotypes also are excluded. Men with the AO genotype, however, can’t be ruled out at this point.

Another genetic marker, the inherited human leukocyte antigen (HLA), is used for paternity testing. If a man and child share the same HLA markers, the probability that the man is the child’s father is 90 percent. When testing combines HLA and ABO typing with another genetic marker known as haptoglobin, the probability approaches 95 percent.

However, DNA matching (see Chapter 15), which offers 99 percent certainty, when properly administered, is the gold standard for assessing paternity.

Checking for semen

At the scene of a sexual assault, the search for semen includes the corpse (in cases of murder) or victim, underwear, condoms, bed sheets and mattresses, carpeting, and flooring. A living victim undergoes a rape exam by a physician. This exam typically takes place in a hospital emergency room, where standardized rape kits are used. Turn to Chapter 12 to find out how investigators handle rape cases.

Tests for semen are either presumptive or confirmatory. Presumptive testing generally is based on the fact that semen contains a very high level of the enzyme acid phosphatase. Confirmatory testing relies on the presence of spermatozoa or prostate-specific antigen (PSA).

Investigators use the following tests to find and analyze semen:

• Presumptive testing: Acid phosphatase enzymes (AP) are a class of proteins that are common in nature and are found in many animals and plants. Semen contains a high level of acid phosphatase, which is produced by the seminal vesicles. This type of AP is called seminal AP or SAP. When SAP is found in a crime-scene fluid sample or stain, it provides presumptive evidence that semen is indeed present. Unfortunately, certain fruit and vegetable juices such as watermelon and cauliflower, some fungi, contraceptive creams, and even vaginal fluid itself can give a false-positive AP test.

  Other presumptive tests search for the presence of two other components of semen: spermine and choline. Each of these tests is positive whenever crystals form after the sample is exposed to certain chemicals.

• Confirmatory testing: If presumptive testing suggests the presence of semen, one or more of the confirmatory tests are then done. The two most commonly used ones are
  • Microscopic examination: Because spermatozoa are present only in semen, finding them is absolute proof that semen is present. A sample is placed on a microscope slide and treated with one of several stains. The viewer typically sees a combination of intact and fragmented spermatozoa. Finding a single sperm or sperm head confirms that the sample is semen.
  • Prostate-specific antigen (PSA): If no spermatozoa are seen, the examiner must resort to testing for prostate-specific antigen (PSA or p30), which is highly concentrated in semen. Testing involves an antigen-antibody reaction that is quick and simple. Finding PSA or p30 confirms the presence of semen. Although a vasectomy may markedly reduce or completely eliminate spermatozoa from the semen, it has no effect on the PSA level, because PSA is produced by the prostate gland, which lies downstream from the site of the vasectomy.

Checking for saliva

Saliva is an important bodily fluid to the forensic examiner. It can be recovered from everything from stamps to food and bite marks. More importantly, it can reveal ABO antigens and thus blood types in secretors and sometimes can yield enough DNA for profiling.

Saliva begins the digestive breakdown of carbohydrates into simple sugars as you chew your food. Amylase is the enzyme that accomplishes this task. Like AP enzymes, amylase enzymes are found in many animals and plants.

Testing for saliva involves testing for the presence of alpha-amylase, the primary amylase found in saliva. No confirmatory tests exist for saliva, only presumptive ones.

Detecting vaginal fluid

Detecting vaginal fluid is difficult, but it may be important in nonejaculatory rapes and penetrations with foreign objects. Swabs can be taken from a suspect’s penis or from any suspected foreign object. Testing depends upon the finding of glycogen-containing epithelial cells. Epithelial cells line the vagina, and glycogen is a starch that is stored within the cells.

Periodic Acid-Schiff (PAS) is a reagent (chemical solution) that stains glycogen a bright magenta color. Whenever glycogen-rich epithelial cells are exposed to PAS, their cytoplasm (the liquid part of the cell) is stained magenta. In cases of object rape, the suspected object is swabbed, and any material obtained from the swab is spread onto a glass slide and then stained with PAS before being viewed under a microscope. Whenever cells with bright magenta staining show up, the material obtained is likely to be vaginal fluid.

The problem is that not all vaginal epithelial cells contain glycogen. Cells from young girls who have not begun menstruating contain none. Similarly, cells from postmenopausal women rarely contain glycogen. The amount of glycogen found in these cells likewise varies with the stages of a woman’s menstrual cycle. As a result, in many cases, the test comes back negative even though vaginal fluids are present.