Testing for Blood

Most presumptive tests for blood are color change tests that are based on the peroxidase- like properties of hemoglobin present in red blood cells (Gaensslen, 1983). Hemoglobin

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will catalyze the oxidation by peroxide of a number of organic compounds to yield colored products. Hydrogen peroxide is most commonly used as the peroxide. These tests can be done directly on the garment or by a transfer technique. A transfer technique is preferred, unless testing extremely small samples or large areas. The Hemastix™ Test (using tetra- methylbenzidine, TMB) is one of the simplest and most sensitive and can be used at scenes as well as on clothing. It consists of a plastic strip with an orange-colored reagent-treated small pad at the end that is rubbed lightly on the suspect bloodstain. The pad is then moist- ened with distilled water. A color change to dark green will occur in the presence of blood. The Kastle-Meyer (KM) test is a two-step test with the reagent phenolphthalein, an acid– base indicator, added to filter paper that has been rubbed on the sample. On addition of an oxidizing agent (hydrogen peroxide in a 3% solution), the transferred sample on the filter paper will turn pink in the presence of blood. Similarly the leucomalachite green (LMG) test produces a green color in the presence of hemoglobin (see James and Nordby, 2005, for presumptive blood tests).

The luminol reagent produces light by undergoing a chemical reaction when it is oxidized in alkaline solution, a process called chemiluminescence (Gaensslen, 1983). Hemoglobin acts as an “accelerator” in these reactions. The luminol test is a presumptive blood test sensitive to 1 in 100,000 dilution (Tobe et al., 2007). It is used predominantly at crime scenes to elucidate bloodstains or blood patterns that are not visible to the naked eye due to dilution, such as in attempts to clean up the scene. The examiner should photograph the luminescence to record the pattern or result. Copper and some other metal salts, as well as plant peroxidases, will give false-positive reactions. It is possible to obtain DNA profiles from latent bloodstains after using the luminol reagent (Manna and Montpetit, 2000). The clothing examiner must observe the item of clothing in darkness, such as an enclosed room in a laboratory with the lights off, because the reaction is light producing.

Bloodstains that have been diluted by machine washing may not be visible, especially on dark-colored clothing, and thus a chemical reagent such as luminol may be the only method for developing latent bloodstains. Luminol can be used to locate latent blood transfer patterns and other larger bloodstain patterns that may have been washed or cleaned up. However, the examiner should use caution when interpreting such diffused or diluted bloodstain patterns

(Adair and Shaw, 2005). Luminol should also not be used to interpret the small blood spatters often created as a result of beating, stabbing, or shooting events (Pex, 2005). The examiner should avoid spraying a chemical directly onto a garment, such as is employed in luminol enhancement, if other techniques can be used. The process alters the remaining stains and deposits, with attendant loss of information, and renders any reexamination difficult.

The ideal presumptive test for blood is one that is specific to blood as much as possible, has a high sensitivity, and will not damage underlying DNA on the garment. Hemastix™, KM reagent, and LMG have been reported to be detected up to a 1 in 10,000 dilution, show false-positives to only a small number of substances other than blood, and achieve DNA amplification even when directly tested on the sample.

Confirmatory tests for blood are crystal tests and immunological tests using antibod- ies. Crystal tests, such as the Takayama test, are simple but may use more of the suspect material than is desired. A sample of the stained area of the garment is placed on a micro- scope slide and covered by a cover slip; a drop of the reagent (such as pyridine hemach- romogen) is added under the cover slip. The sample is then examined using microscopic magnification. If distinctive crystals are produced, then the sample is positive for blood (Gaensslen, 1983).

Immunological confirmatory tests use similar techniques to species identification. Antibodies for various animal bloods are commercially manufactured and are used as a testing chemical for a specific animal antigen. The Ouchterlony double immunodiffusion method (Gaensslen, 1983) involves placing extracts of the bloodstain to be analyzed with specific antisera. If the bloodstain contains antigens corresponding to the specificity of the antisera, the antibodies bind to their antigens and the complex precipitates in a visible line on the gel. Using antibodies specific for human hemoglobin combines the confirmatory test for blood with the human species test into a single procedure.

Recently, new test kits have been marketed as confirmatory tests for human blood (Reynolds, 2004). These kits are especially useful at crime scenes due to their portability but may also be useful in the laboratory for clothing examination. They use an immuno- chromatographic technique that reacts with human hemoglobin.

Many laboratories today perform (1) a presumptive test for blood and then (2) DNA typing on the suspect sample. It is inferred that the sample is of human origin if a profile is obtained, because DNA typing is higher primate specific. A positive presumptive test for blood infers that the sample is human blood. It must be remembered that this is an indirect test; if it is imperative that a sample be confirmed to be blood, then a confirmatory test should be performed. If the size of the sample is an issue, then a sample of the extract used for DNA typing may be used for an Ouchterlony test for human blood.

Although the search for blood on clothing is often straightforward due to the amount of blood shed, in some cases crucial bloodstains have been overlooked due to the small quantity and/or difficulty in discerning the blood on dark clothing. The following case from England illustrates the problems that may face a clothing examiner in a high-profile crime (Rawley and Caddy, 2007):

A 10-year-old boy, Damilola Taylor, died in 2000 on a London housing estate as a result of a stab wound to his thigh. The wound was possibly caused by a broken beer bottle and there was extensive blood loss at the scene. During the first trial of four boys in 2002, no forensic evi- dence existed. Two of the boys were found not guilty, and the charges were dropped against the other two. During a second police investigation, clothing belonging to two brothers, Danny and Rickie Preddie, were submitted for reexamination at a different forensic laboratory from that

Human Biological Evidence 127 which had examined all the original clothing items (more than 400 articles). A white training shoe belonging to Danny Preddie contained a small drop of blood, with directionality from above. This drop was DNA profiled and found to match the profile of Damilola Taylor. The blood drop had not been discovered in the first examination using the naked eye and KM presumptive tests. However, photographs of the shoe from the initial examination showed a “drop” on the shoe. A bloodstain was also found within the ribbing of a cuff of a sleeve of a black windjacket belonging to Rickie Preddie. Again, this stain matched the DNA profile of Damilola Taylor. On the first examination, no blood was detected on the windjacket. A general KM screen had been performed on it, with negative results. On reexamination, reddish-brown particles could be seen under magnification of 40 times on the cuff; these particles were found to be KM positive. The discovery of the two bloodstains led to prosecution of the two brothers, and they were eventually found guilty of manslaughter in 2006.

A government review (Rawley and Caddy, 2007) cited human failure, rather than sys- temic failure, as the reason the two relevant bloodstains were not found in the first examina- tion. The reviewers identified a conflict between the pursuit of excellence and the demand for urgent results. No doubt the large number of clothing exhibits, nearly all marked “urgent,” and the high profile of the case contributed to the pressure of the first examination. This apparent quandary will not surprise the many forensic scientists who are called upon to do work of high quality while under pressure to produce results promptly. A solution could be an impact-based priority system that allows the scientist to focus on the most useful work at the outset but not do everything at once. All clothing examination should be performed using the scientific method and hypothesis testing — that is, formulation of a hypothe- sis and performing a scientific experiment using appropriate methods. Blood from a stab wound to a victim may result in transfer of very small quantities of blood to the clothing of an assailant even though there may be extensive blood loss at the scene; the victim may bleed to death some time after the initial assault, well after the offender has absconded.

7.2 Semen

The presence of semen confirms sexual activity of a specific male; however, it does not determine whether such activity was by consent, nonconsent, or masturbation. The ejacu- late volumes of human males range from 2 to 6 ml and contain a mean value of about 100 million sperm cells per milliliter (Jones, 2005).

The spermatozoa in the semen carry the individualizing traits of male DNA; thus, DNA profiling is generally used to type semen. The head of the sperm contains the cell nucleus, which is packed with DNA. The anterior portion of the head is capped with the acrosome, rich in enzymes to assist in penetrating the cell wall of the female egg during fertilization. A flagellated tail is attached to the head. The tail may be readily separated from the head and is not often seen on dried semen stains on clothing.

Dried semen on clothing may appear as a yellowish-white crust if it is undiluted or in suf- ficient quantity, so it may be readily visible. On white cotton garments, semen stains can have an off-white appearance. They tend to be stiff and turn yellow with age at room temperature.

The most common area for semen sampling in a rape case is the inside crotch of the underpants of the female victim, because this corresponds to vaginal drainage from inter- nal ejaculate. Sometimes there is external ejaculation onto sweaters, pants, jackets, and other clothing items. The victim may also have wiped or spat ejaculate onto clothing, or sat on the edge of a nightgown or tail of a shirt, resulting in drainage.

The location of semen is not as simple as that of blood, because the color and consistency of dried semen can be confused with other body fluid stains. Untreated semen usually shows quite strong fluorescence, and ultraviolet light may locate the stains (see Chapter 3 for assisted light screening techniques), depending on the background luminescence of the clothing material and whether the clothing fabric quenches (absorbs) the fluorescence of the deposits. It should be noted that UV light can, at short wavelengths, degrade DNA, but long-wave UV light has little or no effect on subsequent STR (short tandem repeat) profiling; so examiners should be careful in their choice of light sources. For example, UV light at 254 nm is used to irradiate purchased unused plastic tubes to block DNA replication of any introduced DNA in manufacture. Fluorescence semen detection methods are attractive because they provide a rapid, nondestructive way of locating stains on large items and on a large number of items (Kobus et al., 2002). Because many types of materials, including foods, beverages, and oils, fluoresce, the presence of a fluorescent stain does not confirm semen; nor does a lack of fluo- rescence necessarily exclude the presence of semen, because some fabrics and fabric additives quench fluorescence (absorb the emitted light), so that none is observed.