The Biological Crime Scene It s Not Just About DNA

Transcription

1 The Biological Crime Scene It s Not Just About DNA 13 When asked what his gut feeling was concerning the guilt of a particular suspect, an NYPD detective said, I don t have gut feelings anymore. I wait for DNA * Introduction Consider the totality of the evidence at a crime scene. A mental checklist might include footwear impression evidence, bloodstains, bullets, and so on. All are important, but scene investigators often fail to find one or more, simply assuming that it was not present. This is not unusual and it is expected. For example, ballistics evidence is not expected and should not be present if the crime did not involve a shooting. Failing to locate biological evidence, however, has a different feel because of the lofty importance DNA evidence enjoys. This importance is given to DNA evidence because it can identify someone to the exclusion of everyone in the world. This is why crime scene investigators and scientists often focus on finding biological evidence, so much so that their rallying cry might well be, Find DNA and you ve got your perp. This works because DNA profiles from biological evidence collected at the crime scene are uploaded into an FBI-maintained database CODIS (the Combined DNA Indexing System). Once in the system, scene profiles can be matched to other crime scenes, convicted felons, arrestees, or to identify missing persons. It should not come as a surprise that DNA is so coveted as evidence from both investigational and legal perspectives. There are scenes, however, where investigators fail to find biological evidence. The fact is they missed it. It is probably safe to say that every crime scene involving people probably had biological evidence present, which means that scene investigators have been missing biological evidence for a long time. This statement will cause investigators to bristle, but before dismissing the idea as academic folly, consider it from this perspective: Anyone entering a room brings along something of themselves. When they leave the room, they leave something behind. Certainly, whatever is left behind might be difficult or even impossible to find. Consider this example. An investigator enters a scene and finds a young male body lying on the floor. The deceased has no head hair and a large contusion on the side of his head. There is no obvious bloodstain spatter such as from the impact blow. In fact, there is no apparent active bleeding, only a trickle of blood on the deceased s cheek, no blood droplets on the floor, and no sign of a struggle. Outside the back door and lying on the pavement is an old, broken brick with rough edges. The location is not particularly unusual and should not raise any suspicion that it might be the murder weapon. In fact, picking up the brick and casually examining it reveals nothing except, perhaps, some dirt: certainly no hair and no obvious blood or skin. But this is, indeed, the murder weapon, and biological * Conversation with the author.

2 338 Crime Scene Forensics evidence is present. In fact, by definition, biological evidence must be present. Many scene investigators will fail to collect the brick, and others might, just to be safe. Both may well believe the brick holds no evidentiary value. The forensic scientist in the laboratory would have a different opinion because it is possible to find shed skin cells lodged in the crevices as evidentiary material under a microscope. Certainly, this is not an on-scene procedure, but the message is that biological evidence is always present. Unfortunately, we do not yet have the technology to detect all of its traces at the scene Categories of Biological Evidence The most commonly occurring examples of biological evidence usually come from humans and animals because people are usually the victim or the perpetrator of a crime, and, since people have pets, their biological material may also be present. Figure 13.1 lists examples of forensically important biological evidence, some of which is commonly or not so commonly encountered. In the Common Examples list in Figure 13.1, some examples of forensically important biological evidence, such as blood, occur more often than others. Others, such as fingerprint residue or sloughed-off cells, also occur commonly, but investigators usually do not consider them to be common examples of biological evidence; the former is not thought of as biological evidence and the latter is not usually visible. The reason is that the value of fingerprint residue is thought of in terms of its friction ridge detail. Fingerprints contain biological substances, some of which has current or potential forensic value: fatty acids, proteins, and cells (DNA). Thinking of fingerprint residue a little differently may pave the way for it to become more valuable as an example of biological evidence. What about sloughed-off cells? No one will question the premise that cellular material is an example of biological evidence because it is a source of DNA. So, when a perpetrator holds a weapon or picks up an object with an uncovered hand, cellular material transfers Common Examples Blood Human & Animal Semen Saliva Urine Feces Vomit Hair Fingerprint residue Sloughed Off Cells The Not So Common Examples Bacteria Plant material Pollen Viruses Figure 13.1 Forensically important biological substances.

3 The Biological Crime Scene 339 from the hand to the object. In fact, increasingly modern forensic DNA analysis involves what has become known as touch evidence. This is why scene investigators and scientists must consider all evidence from a biological perspective, even if it is invisible to the naked eye. They must think of evidence differently and consider that biological evidence lurks on unlikely sources. Even the most commonly occurring biological evidence must be considered from the perspective of its location at the scene, its pattern, and its donor Searching for Biological Evidence Arguably the team s most important responsibility, given the critical importance of DNA, is to find and collect anything biological. This is a deceptively easy task; however, because it is everywhere, the most probative of it may represent only a small fraction of the totality of the biological spectrum present. In a very real sense, biological evidence is a critical element of the macroscene bloodstain patterns, droplets, and so on and/or the microscene pollen, bacteria, and so on. With the possible exception of biocrimes (see Chapter 15), the most probative biological evidence comes from people: those who live where the crime occurred, visitors where the crime took place but who had nothing to do with the event, public officials who investigate the scene, and the criminal who commits the crime. The challenge is to find that which is important contained within the milieu of all that is present. This is a huge challenge, and successful searching requires all of one s senses, thinking creatively, and a hefty dose of common sense. Luck helps, too. The successful search for biological evidence, any evidence for that matter, requires special attributes and diligence. Acquiring the appropriate expertise is not a matter of attending a workshop that teaches one how to use an ALS to locate semen or saliva, how to apply presumptive chemical tests at a scene to classify an unknown stain, or how to use immunochromatographic cards to confirm the results of presumptive tests. As Figure 13.2 illustrates, the mission is to Never miss anything probative, which of course, is mission impossible! Categories of cognitive activities necessary to make the scene investigation successful are listed under the second title, The Cognitive Tools. The first line under the title, Your Brain and those that follow, except for The Evidence Cascade, indicate cognitive activities that are almost self-obvious. Certainly, without the appropriate scientific education, experience, brains that think creatively and skeptically, understanding the underlying science behind the technology, and being aware of and knowing how and when to apply it, the scene investigation is poised for disaster. The arrows The cognitive tools Your brain Logical and critical Thinking The evidence analysis cascade Experience Understanding the science and technology Figure 13.2 The scene investigator s mission. pointing from the brain cartoon (in Figure 13.2) to each of these cognitive activities emphasize the need for our best asset, our brain, in ensuring the scene investigation is successful.

4 The evidence analysis cascade (EAC) in Figure 13.3 refers to the analytical sequence used in the crime laboratory to analyze biological evidence. It is, however, a sequence with which crime scene scientists/investigators should be familiar. The EAC is important because it highlights the commonality between what is done in the laboratory and what is done in the field. From a biological perspective, this is critical because using an incorrect approach to locate, collect, and preserve biological evidence can compromise the laboratory s ability to isolate and obtain, say, DNA. The EAC shows the common types of physical evidence (yellow boxes in Figure 13.3) found at the scene that is taken to the laboratory. The left side of the cascade refers to biological evidence. Interestingly, several steps of the EAC have common laboratory and scene equivalents (red boxes in Figure 13.3). The crime laboratory-only categories are in the blue boxes. Those common categories include but are not limited to the following: Observation Presumptive testing Confirmatory testing Pattern analysis Whether done at the scene or in the laboratory, the first stage of any analysis is observation, usually a gross visual inspection, highlighting the evidence using various illumination techniques. The laboratory scientist usually follows the gross inspection using a more rigorous examination, which is not typically something the scene scientist/investigator will do because it is either outside his/her expertise or is not appropriate. However, with the coming of hand-held digital microscopes and magnifying lenses, a form of microscopy might be at the scene. Figure 13.3 The evidence analysis cascade. The Evidence Analysis Cascade Presumptive TestingPattern Analysis Tactile Analysis Species Testing Chemical Testing Immuno Chromat. Gross Visual Examination Biological Evidence Enzyme Testing Confirmatory Testing Genetic Marker Testing Stereomicroscopy Microcrystal Analysis DNA ALS Impression Evidence Pattern Analysis Immuno. Chromat. Lectin analysis Pattern Analysis Trace Evidence Scrapping Tape lift Soil/Paint/Glass Hairs Fibers Confirmatory Testing Instrumental Analysis Microscopy

5 The Biological Crime Scene 341 The next topic is presumptive testing. If blood, semen, or saliva is suspected, a series of presumptive tests can help the scene scientist believe an unknown stain has a biological origin. In-laboratory presumptive tests have typically included touching, chemical testing, enzyme testing, ALS examination, and/or immunological testing. The on-scene version of these tests is essentially the same. The next step in the analytical scheme is confirmatory testing, which would mean using immunochromatographic cards to confirm human blood [1], semen [2], or saliva [3] at the scene. Other confirmatory tests for blood, such as ouchterlony, cross-over electrophoresis, and so on, are laboratory-based and are not applicable for on-scene testing. Laboratory testing of appropriate biological samples typically concludes with DNA analysis. Laboratory and on-scene testing may require a form of pattern analysis and matching. The on-scene and laboratory examinations are similar, but the former differs substantially from the laboratory-based pattern interpretations. For example, bloodstain patterns are best interpreted at the scene because of the importance of viewing them in the context of the events that took place during the commission of the crime. This is, in fact, critical. The laboratory may receive clothing or weapons with bloodstain patterns that must be interpreted, but these interpretations take place outside of the context of the scene environment, though that context cannot be ignored. The message: Crime scene scientists/investigators use many of the same techniques as those used by criminalists in the laboratory. This is why it is important that scene scientists/investigators understand the science behind the testing and the logic of the EAC as an analytical progression Locating Biological Evidence at the Scene As listed in Figure 13.4, our eye is a critical tool for finding biological evidence. Our natural instinct is to think of blood because it is usually visible. But other kinds of biological evidence become visible when technology rescues the eye. Touch is an older technique that was used before ALSs were available. This was a common technique of gently running a gloved or ungloved (an earlier time) hand over the evidence, such as a bed sheet, to find, say, dried semen by its crusty feel. The feel was more intense in the days when latex or nylon gloves were typically not worn. The same feel still exists, but is more or less muted because of the gloves. High-intensity light sources for the most part have replaced touch for locating latent biological stains. The historical technological sequence started with UV lights followed by lasers and then ALSs; the latter make locating biological evidence easier and faster, Locating Biological Evidence The Eye Touch Hi-Intensity & Alternate Light Sources Chemical Tests Enzymatic Tests Immunological Tests Your Brain Figure 13.4 Locating biological evidence at the scene.

6 342 Crime Scene Forensics especially for semen, saliva, urine, and blood. For years, before light sources were commonplace, scene scientists/investigators used chemical tests to determine whether reddish stains might be blood. In the 1950s, they used enzymatic tests (acid phosphatase AP test) to determine whether a crusty stain might be semen. More recently, scene-forward immunological tests have entered the forensic arena, which can confirm whether a stain is human blood, semen, or even saliva. Regardless of advances in technology, the most important tool the scene scientist possesses is a brain, as discussed above and shown in Figure The reason is our brain using critical thinking marries our cognitive activity with technology. It is the best way to find biological evidence. Early scene investigators had few tools and limited technology with which to find biological evidence, which is why they needed their brains, their experience, and knowledge of crime types. The question then, how can one s knowledge of crime types help? Consider homicides, where one expects to find blood. But finding semen at a homicide scene depends on whether the crime was sexually motivated. Finding semen at sexual assault scenes, however, is an expectation as are bite marks and blood. Until DNA evidence became so important, solving burglaries was largely limited to finding the burglar s fingerprints or other physical evidence, such as footwear impressions or blood, coupled with good detective work. If biological evidence was present, such as blood left by the burglar who cut himself, the perpetrator could not be identified because immunological and biochemical genetic testing ABO blood group and enzyme polymorphism testing was not powerful enough. DNA analysis changed that, making it a critical focus of property crime investigations [4]. The discussion that follows focuses on the most common examples of biological evidence, how to find them at the scene, and how to preserve them as evidence for subsequent analysis Commonly Occurring Biological Evidence Blood Unquestionably, blood is the most commonly occurring biological evidence. It is found in most important crime types: homicides, sexual assaults, burglary, assault, and so on. The discussion on blood in Chapter 9 contains the slides: Forensically Speaking: What is Blood and Forensically Critical Information from Blood. The ensuing discussion considered blood and the forensically critical information available from it. It would be appropriate to review that discussion before continuing. The EAC discussed earlier shows the techniques that have been used successfully to find and/or confirm the presence of biological evidence: tactile analysis, chemical testing, enzyme testing, an ALS, and immunochromatographic cards. Historically, tactile analysis and enzyme testing have not played an important role in finding blood at the scene. In modern investigations for blood, the eye, chemical testing, the ALS [5], and immunochromatographic cards form the investigator s arsenal for locating and/or presumptively testing and/or confirming the presence of human blood at the crime scene On-Scene Testing for Blood The term presumptive testing refers to a test result that helps an investigator decide whether a particular stain might have investigative value. A presumptive test, then, is a

7 The Biological Crime Scene 343 maybe test, one where a positive result means that the stain might be blood. These are not confirmation tests. Other tests are necessary to confirm whether biological material is present. As for blood, presumptive tests are available for any forensically important biological material. The following section considers presumptive tests The Unaided Human Eye The oldest presumptive test is the human eye. The lay person or student may think that since blood is red, it is easy to see. Or is it? Actually, dried blood can be red, brown, yellow, green, or black, and understanding the conditions under which these transitions occur is important. The eye, too, is not a stand-alone instrument because it is connected to the brain. So, when we look at something red at the crime scene, it is really our brain that interprets the color and then determines (presumptively) that the red substance is blood. When we make an observation, we are actually evaluating it in the context of our experience, and that is what is being tested. Like eyewitness testimony, however, our experiences are not infallible or applicable to all situations, and certainly it is not the most reliable indicator of the ground truth. For an experienced scene investigator, observing something red having the appearance of blood spatter means that it looks like blood. It does not mean that it is blood. The investigator s experience is important, but his/her certainty is not the test of certitude. Being certain that something is what one thinks it is does not make it so. The human eye, then, is not a confirmatory test, but coupling those observational skills with experience certainly narrows the range of possibilities. Examining evidence with the unaided eye is a good first approach, but technology can enhance the likelihood of finding blood The Aided Human Eye: ALSs Light enhances an investigators ability to see evidence where it normally would be invisible. Oblique lighting is an example of how light helps find impression evidence (see Chapter 11). Although the flashlight remains an important on-scene tool, recent developments in light technology lasers and ALSs have produced portable, high-intensity instruments with tunable wavelengths that can highlight some categories of evidence better. The most useful of these is the ALS, which has proven to be a versatile resource for scene investigators because it enhances the ability of the human eye to see. The molecules that comprise the evidence absorb specific wavelengths of light. When this happens, the evidence will appear dark. If the molecules lose energy, they might be seen as light fluorescence. This happens because an ALS has a tunable wavelength dial that offers the scene scientist choices depending on the scene situation. Tunable wavelengths are typically not available on a normal flashlight. With blood, the ALS has minimal use because no wavelength in the visible spectrum will cause blood to fluoresce. However, there are tricks the scene scientist can use, depending on the surface on which the blood lies. For example, the 415-nm setting on the ALS makes blood appear darker on light backgrounds [5], thus enhancing the apparent visibility. This occurs because dried blood absorbs light at 415 nm, which is why it appears darker instead of reddish or reddish brown. The increase in contrast between the blood and the surface forces the eye into a more favorable region of the electromagnetic spectrum. On a dark surface, the 415-nm setting does not work because making the blood appear darker is counterproductive. Figure 13.5 illustrates the point. Workshop VII-1 also illustrates this point.

8 Table 13.5 ALS Wavelengths used to locate biological and other evidence Wavelength (nm) Principle Use White Light General scene scanning Fingerprints on shiny surfaces Blood on dark shiny surfaces Long Wavelength General screening UV: Hair, fiber, fluorescent materialpowders Body fluids/bruises/bite marks/tomato-based foodstuff Other Food stains 415 Darkens Bloodstains Can darken tomato based food stains 450 General screening Body Fluids (semen/saliva/urine) fluorescent materials Viewing Goggles Clear Clear or Yellow Clear Orange Powders/Teeth/Bones 465 Fluorescent materials/powders Orange Body fluids 525/570 Super Glue Prints Red

9 345 Crime Scene Forensics Blood on dark surfaces is difficult to see and is easily missed, which forces scene scientists/investigators to choose alternate methods. The first and easiest is to subtract out the background by using different wavelengths (colors) on the ALS. If successful, the blood will usually appear dark against a lighter background. An example could be dried blood on a red wall. Here, the contrast between the blood and the wall is minimal. The CSS setting on the CrimeScope TM 400 (blue-green) on the ALS can lighten the background (light blue) without affecting the darker color of the blood. The result will be dark blood spots on a light blue background. Reddish or dark foodstuffs stain can be confused with blood, so differentiating these from blood prevents the crime laboratory from having to analyze superfluous and irrelevant evidence. Such differentiation is easily accomplished using the ALS: Blood absorbs light at 415 nm and will not fluoresce under long-wave UV light ( nm settings on an ALS). Tomato-based foods may or may not absorb light at 415 nm (usually less so than blood) but usually give a blue-white fluorescence and a yellow or yellow-orange fluorescence under long-wave UV light. Figure 13.5 is an example of blood and ketchup smeared on an orange wall, allowed to dry, then photographed under long-wave UV light from an ALS, and observed using clear goggles. The stain on the left left arrow is a bloodstain and the stain on the right right arrow is a ketchup stain. The blood absorbs the light, which is why it appears darker. The ketchup has a blue-white fluorescence. On dark, shiny surfaces, such as shiny magazine covers, the ALS helps little in subtracting out the background. In these instances, however, oblique lighting using the whitelight setting on the ALS or a simple flashlight is useful. Table 13.1 gives examples of ALS wavelengths and how they can be used to highlight/locate biological evidence at the scene. The right column lists what goggles to use IR Cameras: Combining Searching and Photography Fortunately for scene investigators blood absorbs light in the IR region, which makes it appear dark, much like it absorbs light at 415 nm. In certain situations, this can be useful Figure 13.5 Using the alternate light source to identify blood. Table 13.1 Wavelengths of Alternate Light Source to Locate Biological Material to help visualize blood on dark backgrounds or, sometimes, on dark, shiny surfaces as well.

10 In the past, using IR light to locate blood was a delayed process because developing IR film was necessary. This made on-scene usefulness suspect because it took time before the investigators knew whether the IR light had found blood. Usually, IR photography was used as a vehicle to highlight blood patterns on dark surfaces where it was known that blood was present and was not an on-scene mechanism for finding difficult-to-see bloodstains. The digital IR camera and the ALS have become valuable on-scene partners as tools to help locate dried blood on difficult surfaces. The reason is that digital cameras have characteristics different from those of film cameras simply because the LCD viewers in the former allow scene scientists/investigators to see the blood in situ without having to take a photograph and waiting for film to be developed. Now, there is an instantaneous peek at what is present on the dark surface that does not absorb light in the IR region of the electromagnetic spectrum. In essence, the camera expands an investigator s sight range into the real-time near IR, making the digital IR camera an indispensible tool for on-scene investigations when the ALS is of little or no help. Importantly, too, the LCD IR image can be photographed and included in the crime scene unit s case file. The IR-highlighted stain can be tested with presumptive chemicals or tested using immunochromatographic cards to ascertain whether it is blood or human blood, respectively. Figure 13.6 shows a photograph of the LCD viewer of an IR camera (Fuji 9000S converted IR mode) with an image of a red carpet. The stains on the carpet were invisible to the naked eye and to the settings on the ALS the ALS could not effectively subtract out the background to visualize the blood. The stains, however, were visible in the LCD on the IR camera. Unfortunately, all dark or reddish surfaces are not amenable to the digital IR technique. In order for the IR principle described to work, the surface must not absorb light in the IR region. If it does, both the blood and the surface will appear dark. It is simple enough to find whether the surface absorbs light. Simply turn on the digital IR camera and look through the LCD viewer. If the surface appears dark, it absorbs light in the IR region. If it appears whitish, it does not, and blood should be visible if it is present.

11 347 Crime Scene Forensics Figure 13.6 Photograph of liquid crystal display of infrared digital camera Chemical Presumptive Testing Chemical tests that react with blood were developed in the mid-nineteenth century. Their importance is to give investigators a method of determining whether an unknown stain might be blood by narrowing the range of possible substances by approximately 95%. A positive chemical test means that there is approximately a 95% chance that the unknown stain is or contains blood. This is important because many reddish or dark stains at a crime scene are not blood. A simple example is a stain made from the spray of a shaken Coca Cola TM can on a dark wall. Each of these chemical tests works on the same principle. A method for using these reagents at a crime scene is shown in the biological Workshop VII-1. The chemicals used to identify a stain as possibly being blood can be divided into two categories: those that produce colors and those that produce luminescence [6,7]. The former include a range of dyes that turn color in the presence of hemoglobin, a protein component of blood, and peroxide hydrogen peroxide is the most commonly used peroxide. Vast arrays of these reagents are available commercially. The most common include: phenolphthalein (KM reagents), leucocrystal violet (LCV), TMB, ortho toluidine, ortho tolidine, and leucomalachite green, among others. The second category includes chemicals that also react with hemoglobin and peroxide but instead of turning color, they luminesce, known as chemiluminescence. This group includes luminol, the chemiluminescence of which has been extensively investigated [8 10]. BlueStar TM and fluorescein is used primarily at crime scenes where clean-up is suspected. Luminol and fluorescein have enjoyed a long forensic history, but BlueStar TM is a recently available formulation for which claims of greater and longer luminescent intensity exist. Although it might seem as though BlueStar TM is a new reagent, it is apparently an optimized and reformulated version of luminol. Claims are that BlueStar TM is superior to luminol for crime scene work for several reasons. One important reason is that its luminescence can be seen in dim instead of dark areas [11]. BlueStar TM comes in two formulations, one for on-scene use and the other for training. Since the reagent is expensive, the training formulation is a less expensive version but its manufacturers warn it will destroy DNA. The more expensive, nontraining version of BlueStar TM designed for onscene use supposedly does not destroy DNA. Certainly the luminescence produced can be

12 The Biological Crime Scene 349 Figure 13.7 Bluestar enhancement of washed bloodstains. (Photograph by Robert C. Shaler.) dramatic as shown in Figures 13.7 and The sink in the photograph had been washed with water as had the red wall. Blood was not visible before students began working on the scene. These chemicals work on the same principle. Called catalytic tests, they have been around for over 100 years. The general reaction is shown in Figure The heme moiety of hemoglobin (the red protein in blood) reacts with and cleaves hydrogen peroxide producing an oxygen free radical step 1 in Figure The free radical reacts with a dye (the reduced version) in step 2, oxidizing it to produce a color. The color formed depends on the chemical properties of the dye, with colors ranging from pink to intense green. Figure illustrates the process for one of the more common blood presumptive catalytic reagents the KM reagent [12]. The step-wise process described is for absorbing blood onto a moistened sterile cotton swab. The subsequent steps to administer the test are common for all presumptive tests except for luminol and BlueStar TM, which produce a luminescent product instead of a colored one. For the luminescence-producing reagents, the room must be darkened and the blood fixed (so that it does not dissolve) by spraying with a 2% solution of 5-sulfosalicylic acid. During a long photographic exposure, the area is sprayed with luminol or BlueStar TM. The luminescence will fade, so it is critical to document the reaction photographically (Figures 13.7 and 13.8), and not merely to record in notes that a positive luminol (BlueStar TM ) Figure 13.8 Bluestar developed bloodstains on red wall. (Photograph by Robert C. Shaler.)

13 The Biological Crime Scene 349 Blood: Chemical Presumptive tests General Considerations Step 1: Oxygen free radicals cleaved from peroxide group Heme Fe+++ 2H 2 O 2 Heme Fe++ 2O + 2H 2 O Step 2: Oxygen free radicals react with reduced dye O + Chemical reduced chemical oxidized Presumptive test detects oxidized organic dyes Figure 13.9 General reaction of blood presumptive chemical tests. reaction was obtained. There are two important photographic requirements: (i) The scene details (e.g., furniture, chairs, carpets, etc.) should be visible in the photograph. and (ii) the luminescence should overlay the scene so that the scene details are visible. Examples are shown in Figures 13.7 and 13.8 in the context of the room (red wall) and on the sink in the bathroom. The general steps for accomplishing this are listed below. Darken the room (if using luminal or dim the room for BlueStar TM ) or area as much as possible. Even with BlueStar TM, the darker the better. Sometimes covering windows, door areas, exit lights, etc., with black plastic bags will suffice. Spray the suspect area with 2% 5-sulfosalicylic acid and allow it to dry. Set the camera on a tripod, set the aperture to bulb, turn off lights, and take photograph of the area scene using a 2-min exposure. Check that photograph is not overexposed. If overexposed, adjust the shutter speed and retake the photograph. If the exposure is adequate (i.e., scene detail is visible in the photograph), trip the shutter and spray the area with BlueStar TM. Allow luminescence to develop. When Commonly used presumptive test for blood Ensure no excess of water Disssolve stain onto tip of swab Add drop of ethanol Cotton swab Add drop of KM reagent to stain color change at this point: false positive Add 3% H2O2 Observe pink color KM positive Figure Kastle-Meyer reaction with dried blood.

14 350 Crime Scene Forensics fluorescence begins to fade, spray the area again. Continue this process for the entire 2 min. Then trip the shutter and observe the photograph. False positive luminescence is possible with luminol and BlueStar TM, usually with metals and bleach. Since bleach is used to clean blood, the bleach-cleaned areas give an initial luminescence that fades quickly. The same is true for certain metals, for example, copper pipes, and so on. Presumptive blood testing reagents are useful because they provide immediate investigative information. Substances that have peroxidase activity, such as horseradish, also give a false positive. The choice of which to use and when is important. For example, consider the scene in which an informant says that a particular individual had been murdered there years earlier. Certainly, finding the blood visually or even with an ALS might be fruitless. It might even be that the entire scene had been remodeled or repainted. The team leader will need to decide how to approach the problem. After an exhaustive but unsuccessful search for visible blood, the team leader might discuss the following with the team: Should the team spray using BlueStar TM and, if so, can the team darken the room sufficiently? Should the team use a reagent that forms a color, such as leucocrystal violet? [13] If the investigation is based on an informant s information that the room had, for example, been painted to hide blood, the team must consider the possibility of finding the blood under the paint and discuss how to accomplish that. Certainly, one consideration is spraying with BlueStar TM Lateral Flow Immunochromatography Lateral flow immunochromatography is a rapid technique for identifying small amounts of specific molecules. Its forensic application has been largely used to identify forensically important biological substances blood, semen, saliva, and urine. The specific tests can be conducted at the scene; however, unlike the presumptive chemical testing reagents discussed above, they must be purchased commercially, which raises the cost per test significantly. Unlike presumptive tests, they specifically identify unknown stains as human blood (some cards cross-react with ferret blood), semen, or saliva (salivary amylase). Although these tests come in the form of testing kits, they, too, must pass quality tests for sensitivity and specificity. The same set of cotton swabs prepared above (assuming that human fluids are used) for blood will suffice a separate set of cotton swabs should each be prepared for human semen and saliva. A tabular record, such as Table 13.1, should be maintained with other quality records. A forensic validation application of these immunochromatographic cards has been published [14]. A problem with immunochromatographic cards (rapid stain identification RSID cards), especially those from Abacus Diagnostics and OTEB, is that they suffer from what is known as the hook effect [15]. This happens when testing overly concentrated samples of human blood (RSID cards do not demonstrate a hook effect). The result is a negative test, even if human blood is present. If the scene scientist is not aware and does not understand this anomaly and fails to test an appropriately small or diluted human blood, an incorrect and potentially misleading result will be obtained. When the hook effect occurs, the sample must be diluted and rerun. The quickness, ease of operation, specificity, and sensitivity K12738_C013.indd 350 7/2/2011 3:28:41 AM

15 351 Crime Scene Forensics of these immunochromatographic cards makes it tempting to avoid the traditional chemical tests entirely. If cost is not an issue, this might be the best choice because these tests confirm the presence of human blood in a single test. Importantly, the used immunocard and/or the extract used to run the test can be submitted to the laboratory for DNA analysis, which would save the laboratory time in selecting certain scene stains from submitted crime scene samples for DNA analysis* [16], although laboratories normally prefer to extract the samples in-house. The stain extract used for the cards at the scene should also be saved because the DNA can be analyzed. Cost aside, for many scenes using the immunochromatographic cards exclusively is probably a mistake, if all blood tested turns out to be nonhuman. A negative test typically means that human blood is not present. As mentioned, false negatives with highly concentrated blood extracts can be obtained because of the hook effect. One exception is the blood test by RSID which has no hook effect [17]. A true negative occurs when there is insufficient human blood present; all tests have limits of sensitivity. Of the immunocards available, the Abacus Diagnostics card for blood is the more sensitive; the HemaTrace TM card detects lower amounts of blood than the RSID card. Scene practitioners should be aware of the difference between products among manufacturers and should test the cards for sensitivity and specificity as part of the validation aspect of a comprehensive quality assurance program Collecting and Packaging Blood Evidence The mechanism used to collect and preserve blood evidence is critical. If done incorrectly, the result can compromise or destroy the evidence. There are seven invariant rules: Always wear protective clothing face masks, gloves, proper outer wear, and shoe/ boot covers. Dry all blood evidence. If that is not possible, transport it to the forensic laboratory as soon as possible, ensuring that it does not come into contact with other evidence. Never package blood evidence in plastic. Always use paper. Package each item of evidence individually. Never mix items. Never allow adjacent bloodstains (even if dry) to come into contact with other stains on the same or other items of evidence. Store all biological evidence in a cool dark place, if possible. This means keeping the evidence away from sunlight and heat, such as in a police car or crime scene unit vehicle on a hot day, while processing the scene. Collect the entire sample, if possible. This may not be possible because of the size or fixed location of an object. If this is not possible, cut the entire stain from the larger item and document the location photographically. If necessary, collect evidence on lightly moistened swabs. Following these rules ensures that blood evidence biological evidence generally is collected and preserved properly. * Reena Roy and Tam Ho, personal communication.

16 The Biological Crime Scene 351 Blood evidence comes in forms and can occur in patterns that have important interpretative information. Collecting it properly depends on the form it takes: pools, droplets, trail swipes, castoff, weapons, furniture, and clothing. If possible, the entire bloody object should be collected and packaged. This gives the laboratory the opportunity to decide which stains are important. If that decision is made at the scene, there is the real possibility that important evidence might be lost. For example, if a grouping of dried blood stains on a piece of furniture has more than one individual s blood present and if the investigator chooses only one of several stains to collect, the second person s blood might never be analyzed, which can compromise the scene analysis, an understanding of the ground truth of w h a t h a p p e n e d, a n d t h u s t h e r e c o n s t r u c t i o n o f t h e e v e n t s. If the blood evidence is wet, say, a droplet lying on a tile foyer, the following steps should be followed: Determine the length time it takes to dry (carefully monitor how long it takes to dry). Collect it by scrapping it into an appropriate collection vehicle a druggist fold bindle or coin or glassine envelope. Record the temperature, humidity, and the conditions where the stain was located (windy, sunlight, shade, etc). If the stain is large, such as a wet pool, the following steps should be followed: Absorb a small amount of the stain onto a cotton swab. Place it into a small swab box (a small rectangular box with holes near one end like those used to dry vaginal swabs in sexual assault cases) Allow it to dry. If the evidence is a blood trail, the following steps should be followed: Determine the direction of travel. Photograph the diagnostic droplets to preserve the information. Collect representative droplets from the beginning, middle, and end of the trail by swabbing them onto a moistened cotton swab or scrapping them into separate envelopes. Package them separately. A stepwise process for swabbing is shown below: Lightly moisten swab and shake off excess. Dissolve bloodstain onto tip of swab, keeping sample as concentrated as possible. Dry swab appropriately, ideally in swab drying box. Package dried swab (wet swab cannot be dried immediately) in swab box. Dried blood found on clothing should be packaged by folding the garment around brown or white wrapping paper, ensuring that individual stains on the garment do not come into contact with other stains. After folding the evidence, the entire garment should be placed in a separate paper evidence bag and labeled appropriately Semen The second most prominent class of biological evidence found at scenes is semen. Generally, modern scene scientists/investigators locate semen using an ALS on the CSS setting or

17 353 Crime Scene Forensics using another appropriate wavelength that causes semen to fluoresce. Typically, the scene scientist/investigator wears orange goggles using blue light at approximately 450 nm, but the wavelength is what dictates whether to use orange or some other color goggles, if any. For some wavelengths, for example, 415 nm, only clear goggles are necessary. Every investigator knows what semen is, but few have a complete understanding of its biological origin or its forensic potential. The male ejaculate is a liquid that contains cells, the origin of which is mostly the seminal vesicles approximately 46% 80%, with the prostate gland producing between 13% and 33%; the remainder comes from the testicles, epididymus, and the bulbourethral and urethral glands. All forensic investigators also know that semen plays an important role in identifying assailants in sexually motivated crimes because of the DNA present in the spermatozoa. Like blood, semen has a much broader forensic potential than simply as a source of DNA. Students and novice investigators should understand how broad this potential can be. Figure illustrates this. Like blood, semen has cellular and liquid (plasma) fractions. Each potentially can play an important forensic role. For example, the liquid portion, seminal plasma, contains the same soluble components found in blood. The same forensic information is potentially available. For example, metabolites of illicit and therapeutic drugs are present which are identifiable in the semen removed from the vaginal swabs of a rape survivor. If the DNA profile from the spermatozoa cannot be matched to a previously incarcerated felon in CODIS, it has little investigative value. However, learning that the semen has an illicit drug profile may provide important investigative leads, such as people to interrogate. Likewise, a smoker has nicotine metabolites present, which gives personal information about the assailant. Like blood analysis, semen analysis has evolved in parallel with advances in science. Since the 1600s, when Antonie van Leeuwenhoek s student first saw a spermatozoan in the microscope, identifying semen microscopically has been a forensic laboratory s gold standard. Forensic laboratory scientists, though, work with dried stains, so a microscopic analysis, while necessary, is time consuming. In the early part of the twentieth century, much of the semen identification in rape cases was done in hospital laboratories, which Semen complex connective tissue Hormones Salts Drugs Antigens Enzymes Plasma Genetic markers Antibodies Blood group substances Isoenzymes Individual specific antibodies White cells HLA antigens DNA Cells Sperm cells Genetic markers Pre-sperm cells Blood group antigens Isoenzymes received a Figure Forensically speaking, what is semen?

18 The Biological Crime Scene 355 vaginal lavage as part of the clinical examination of the sexual assault survivor. Generally, hospital laboratories performed two tests: a sperm mobility test and a quantitative AP or prostatic acid phosphatase (PAP) analysis, the latter popular in the 1940s, 1950s, and 1960s as a way to show that semen was present in the vaginal vault. The analysis of AP (or PAP) gained popularity because semen has high concentrations of it [18,19]. Hospital laboratories quantified the amount of AP present. If the level was above a certain cut-off level, the interpretation was that semen was present, which would be diagnostic of sexual penetration. Forensic laboratories went in a different analytical and interpretive direction. They typically received dried vaginal swabs from the police instead of a vaginal lavage, which made determining the level of AP difficult. Thus, they used the AP test differently. They added a small amount of the AP reagent to a small cutting of the dried stain and measured the time it took for the test reagent to turn color. If the color change was rapid, less than 30 s, usually almost instantaneously unless the sample was very old, the laboratory had evidence that the dried stain might contain semen. In essence, the AP test was used as a presumptive indication that semen might be present. They confirmed the presence of semen microscopically by observing spermatozoa. The purpose of this discussion is to show that forensic scientists have historically used a sequence of tests to identify semen in dried stains, culminating in identifying the mechanism of conception, spermatozoa. This analytical sequence changed in the 1970s with the discovery of a semen-specific protein known as p30 by forensic scientists [20,21] and prostate specific antigen (PSA) by clinical scientists [22]. For on-scene applications, there has historically neither been the time nor the resources to perform microscopic or immunological analyses for spermatozoa. In fact, that is not the job of the scene investigator. They are responsible for finding the evidence and transporting it to a forensic laboratory for analysis. In the 1950s and 1960s, the crusty feel of dried semen was a quick check for the presence of semen. At that time, laboratory scientists used this same tactile test because it was fast and could be easily performed on evidence where semen might logically be expected, such as bed sheets. Another on-scene technique was the use of the first true ALS, the black light also known as Wood s light [23]. Short-wave UV light caused semen to fluoresce, and it worked well for semen stains on light, unwashed, as well as dark fabric. It did not work on light, detergent-washed fabric because detergents in the fabric interfered with the luminescence of the semen. The same AP test as used in the forensic laboratory to presumptively identify semen was adapted for scene use. The test was used essentially like the chemical tests for blood. Interpreting it was much the same, too, and was based on the time it took to produce a specific color. It was and still is a presumptive test. A schematic of the components of the AP test is shown in Figure Performing the AP Test on Fresh Semen Stains Quality Assurance and Control Testing On the day of use, a known semen stain (positive control) and a negative reagent control (distilled water) are tested to ensure the reagents meet minimum standards. General guidelines for ensuring the quality of testing reagents are discussed in Section All quality results must be archived. If controls fail, it is poor practice to continue using the reagent until the problem has been resolved or new reagents that meet minimum standards are prepared. If the results of the test are positive, a substrate control (if available)

19 Acid phosphatase o-dianisidine The Biological Crime Scene 355 Semen presumptive test Acid phosphatase (AP) test Sodium alpha-naphthyl phosphate Alpha-naphthyl phosphate Molecule split by AP Frees the naphthyl group Fast blue ortho dianisidine Combines with naphthyl group Produces bright pink to a dark blue/purple color Blue-purple color Figure Acid phosphatase test for semen. must also be tested, unless the stain is on a cotton swab. The test results must be maintained in the case file. AP test procedure Lightly moisten sterile swab with distilled water and press or gently rub it against the suspected stain. Add one to two drops of sodium a-naphthyl AP solution. Add one to two drops of o-dianisidine dye solution. The development of a blue/purple color within s is indicative of AP levels consistent with semen. For samples giving inconclusive result (longer color development times), semen must be confirmed microscopically or by showing the presence of p30. Interpretation of results Positive reaction: Blue/purple color within s. Negative reaction: No color development, slight/slow color development. Inconclusive reaction: Slow moderate to strong color development longer than 15 s and not longer than 30 s. There are variations on how to run the AP test. One is to dab the unknown stain with a cotton swab moistened with the reagent and wait for the color to change on the evidence or the moistened swab. This is not a preferred method. A more preferable method is to moisten the swab with water, dissolve a small amount of the unknown stain onto the swab, and then test the swab with the AP reagent. A rapid color change from colorless to dark blue to purple is a positive test. Another, but not recommended, variation is to moisten the unknown stain directly with the reagent by dropping microdroplets from a micropipette directly onto the stain. Again, a rapid color change is diagnostic of a positive test. Another variation is to screen large areas to locate invisible semen stains, a technique called AP screening. A piece of filter paper is moistened with water and then rubbed over larger areas of, say, a bed sheet sectioned into a grid. A positive AP test result on the filter paper is an indication that semen is present in that grid area. The AP screening method is not as popular since the introduction of high-powered K12738_C013.indd 355 7/2/2011 3:28:42 AM

20 The Biological Crime Scene 355 ALSs, which highlight semen and other biological fluids better and faster without the use of chemicals. Confirmatory tests were not available for on-scene dried semen testing until recently. The immunochromatographic cards described for blood are also available for on-scene testing to identify the presence of semen proteins, p30 (also known as PSA, manufactured by Abacus Diagnostics) [24], and semenogelin manufactured by RSID [25]. The proper use of the cards can quickly identify human semen at the scene. The AP test, which is still useful, does not identify human semen or even semen. It is a presumptive test. Immunochromatographic cards are expensive, which means the relatively inexpensive AP test, which requires fresh reagents, can screen unknown stains quicker and cheaper. The ALS is superior because, except for the initial expense, it covers a larger area and requires no testing reagents Establishing a Rationale for Searching for Semen A rationale or decision tree should be in place to determine how to go about locating biological evidence at a scene. Certainly, the first line of attack is logic, which leads to where semen might be. At obviously sexually motivated crimes, searching for semen takes place in the most logical places: beds, sofas, car seats, and so on. However, there are nonsexually motivated scenes where a perpetrator might masturbate, urinate, or defecate. In fact, burglary scenes have a higher incidence of semen than one might expect. For whatever reason, perhaps after fondling a female s undergarments or jewelry, burglars masturbate and inadvertently leave their biological signature. So searching for semen should not be confined to the obvious crime type and should not be a one-step, check-the-bedroom-only endeavor. There is still the question of how an investigator should search for semen evidence. The easiest and probably the best method is using the ALS, a first line of attack. Even when using the ALS, there should be a rationale, perhaps a written procedure, for deciding which ALS positive stains to test further and how and which to collect for the laboratory. A tabular record should be maintained of which wavelengths to use with photographs of what to expect. These should be prepared by the investigating unit, as photographs are guides but not as good as hand-on experience. It might seem surprising but there are alternative approaches. In each of the following approaches, the ALS is the first avenue of analysis, but it should never be forgotten that the ALS is useful for locating not only semen but also other biological evidence, such as saliva, urine, and trace evidence. Use the ALS to locate possible semen, saliva, or urine stains and allow the laboratory to decide which to test further. This is by far the safest approach because it leaves the choice of subsequent DNA analysis to the laboratory. Mark and photograph any stains that the ALS highlights. Use the ALS to locate possible probative stains and then use the AP test to determine which might be semen. The laboratory would likely retest the stain with the ALS and the AP test and then perform DNA analysis, if warranted. Procedures for using the ALS, the AP (also discussed above) test, and immunochromatographic cards for semen testing are presented in Workshop VII-3. Use the ALS followed by an immunochromatographic test (the AP test is not used). The more definitive/confirmatory immunological test is performed. A positive test shows the stain analyzed is semen, which alerts the laboratory that subsequent analysis is warranted. However, a negative immunochromatographic card test