Lecture 23 The Biological Crime Scene It’s Not Just About DNA

1. Lecture 23 The Biological Crime Scene It’s Not Just About DNA Locard: "Every Contact Leaves a Trace". Blood at the Scene is the most visible example of the Locard Exchange Principle

2. Biological Evidence • Think about the totality of evidence one might expect at a crime scene: • impression evidence, • bloodstains, • bullets, etc. • Trace evidence • All are important, • Scene investigators often fail to find one or more, simply assuming not present. • Not unusual. It is expected because of the crime circumstances. • 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 stature of DNA. BECAUSE it can identify someone. • Crime scene investigators and scientists focus FIRST 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 (Combined DNA Indexing System). CODIS: Once in the system, scene profiles can be matched to other crime scenes, convicted felons, arrestees or to identify missing persons. • DNA is coveted as evidence from both investigational and legal perspectives.

3. How Much Biological Evidence Is at a Crime Scene? • At some scenes, investigators fail to find biological evidence. • It is probably safe to say that every crime scene involving people probably had biological evidence … means that investigators have been missing biological evidence for a long time. • Reality: In homicide cases: Only 5-10% yield of biological evidence • The fact is, they missed it. • Consider this • Anyone entering a room brings something of themselves with them. When they leave the room, they leave something behind. Certainly, whatever is left behind might be difficult or even impossible to find. • But new technology on the nebulous horizon might be able to find it.

4. Example Case: • Investigator finds young deceased male on the floor. no head hair and a large contusion on the side of his head. • No obvious bloodstain impact spatter. • No apparent active bleeding: only a trickle of blood on the deceased’s cheek, • No blood droplets on the floor … no sign of a struggle. • Outside the back door on the pavement is an old, broken brick with rough edges. … Location is not particularly unusual … should not any raise suspicion that it might be the murder weapon. • Picking up the brick and casually examining it reveals nothing except, perhaps, some dirt: certainly no hair and there is no obvious blood or skin. • But this is the murder weapon AND biological evidence is present. By definition, biological evidence must be present. Many scene investigators will fail to collect the brick, and others might … just to be safe. • Both investigators might believe the brick holds no evidentiary value. The forensic scientist in the laboratory could have a different opinion based on finding shed skin cells lodged in the its crevices, • A technique that has not yet found its way into the field. • The message: • Biological evidence is always present. • Unfortunately, we do not yet have the technology to detect all of its traces

5. What is Biological Evidence Common Examples Blood – Human & Animal Semen Saliva Urine Feces Vomit Hair Fingerprint residue Sloughed Off Cells Not So Common Examples Bacteria Plant material Pollen Viruses • “Common Examples” … examples of forensically important biological evidence occur more than others. • Others also occur commonly … fingerprint residue or sloughed off cells … investigators do not think of them as common biological evidence; • Fingerprints: not thought of as biological evid • Sloughed-off Cells: not 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.

6. Finding Biological Evidence • Critical elements of the macro and micro scenes • Bloodstain patterns, droplets, etc • Pollen, bacteria, etc. • Arguably leader’s most important responsibility, given the critical importance of DNA, is to find and collect anything having biological properties. This is a top priority. • Not an easy task: • Is it everywhere • Represents only a small fraction of the totality of the biological spectrum present. • With the possible exception of a bio-terrorist event, the most probative biological evidence comes from people. • Who live where the crime occurred, • visitors where the crime took place but had nothing to do with the event, • public officials who investigate the scene and the • criminal who commits the crime. • Challenge: Find important probative biological evidence among all present. • Successful searching requires : All senses, ability to think creatively, common sense … Luck, too.

7. The Cognitive Tools • The Evidence Analysis Cascade • Your Brain • Logical & Critical Thinking • Experience • Understanding the Science and Technology The investigator’s mission is “Never Miss Anything Probative” • The Intellectual Approach is the Key to Success • 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 technology … the scene investigation is poised for disaster. • 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 how to use an ALS to locate evidence: semen, saliva or urine or how to employ presumptive chemical tests to classify an unknown stain as blood.

8. Locating Critical Biological Evidence • Historically: • Blood: Chemical tests commonplace 100 years ago to determine whether reddish stains might be blood. • Semen: In the 1950’s they used enzymatic tests (acid phosphatase test) to determine whether a crusty stain might be semen. • Modern Techniques use technology • 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 alternate light sources (ALS). • ALS’s, make locating biological evidence easier, especially semen, saliva, urine and blood. • 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 the brain … critical thinking (the brain) marries technology. • Brain is best and only way to find biological evidence. • Eye • Brain • Touch • Hi-Intensity light • ALS • Chemical Tests • Enzymatic Tests • Immunological Tests

9. The Scene & the Forensic Laboratory The Evidence Analysis Cascade

10. Red = Common Techniques Blue = Lab only Techniques

11. Commonly Occurring Biological Evidence

12. Blood

13. What Is Blood? Blood Cells Liquid Serum Plasma Red Blood Cells White Blood Cells

14. Forensically Speaking: What is Blood? Blood Complex Connective Tissue Plasma Cells Hormones Antigens White Cells Red Cells Salts Drugs Antibodies Genetic Markers Enzymes Blood Group Substances HLA Antigens Blood Group Antigens Individual Specific Antibodies DNA Isoenzymes

15. Forensically Critical Information from Blood Drugs of Abuse Prescriptions Genetic Markers Psychological Behavior Identification Disease Susceptibility Ancestry Sexing Racial Identity Individual Identity DNA Profiling Antibody Profiling

16. On-scene testing for blood Could It Be Blood? • Presumptive testing … • A test helps an investigator decide whether a particular stain MIGHT be blood and thus have investigative value. • A presumptive test, then, is a “maybe” test, one where a positive result means that the stain might be blood. These are not confirmation tests. • Other tests are needed to confirm whether the biological material is present.

17. The Unaided Human Eye • The eye: • Oldest presumptive test. • Not always red. • Dried blood can be red, brown, yellow, green or black, • Understanding the conditions under which these transitions occur is important. • The eye, not a stand-alone-instrument because connected to brain. • Interprets color and then determines (presumptively) that red substance is blood. • Evaluating it in the context of our experience, a • Experience is what is really what is being tested. • Not a confirmatory test, • Coupling observational skills with experience narrows the range of possibilities. • A good first approach, but technology can enhance the likelihood of finding blood. • Experiences are not infallible or applicable to all situations, and 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. • Does not mean that it is blood. • Certainly the investigator’s experience is important, but certainty is not the test of certitude. • Being “certain” that something is what one thinks it is does not make it so.

18. The Aided Human Eye – Alternate Light Sources (ALS) • Light enhances ability to “see” evidence where it normally would be invisible. Oblique lighting is an example of how light helps find impression evidence. • Flashlight is an important on-scene tool, • Recent developments in light technology – lasers and alternate light sources – have produced portable, high intensity instruments with tunable wavelengths that can highlight some categories of evidence better. • Useful example is ALS, which has proven to be a versatile resource for scene investigators because it enhances the ability of the human eye to “see” better. • 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 is a topic we discussed in Part I of this lecture series. 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.

19. Using Light SourcesTo Find Biological Evidence

20. The Electromagnetic Spectrum Using Light to Find Biological Evidence Ultraviolet Region 190-290 290-400 Short wave Long wave Visible Region 400-455 455-492 492-577 577-597 597-622 622-700 Violet Blue Green Yellow Orange Red Infrared Region Blood Absorbs Light Appears Dark >700 IR

21. Detecting Blood with the ALS Blood on Light Colored Surfaces • “Tricks”, depending on the surface on which the blood lies. • For example, the 415nm (and 450nm) setting on the ALS (violet light) makes the blood appear darker on light backgrounds, • Enhances apparent visibility. • Dried blood absorbs light at that 415nm, • 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.

22. Blood on dark surfaces • Dark Surfaces • 415nm approach does not work • Making the blood appear darker is counterproductive because the contrast between the surface and the blood is diminished • Difficult to see and easily missed, … forces scene investigators to choose alternative methods. • Subtract background: • Use light of different wavelengths (colors) … ALS. • If successful, blood will appear dark against a lighter background. • An example is blood on a red wall. • Oblique lighting on shiny surfaces • IR light

23. Differentiating Food Stains From Blood Long-wave UV Light On Wall • Differentiating food from blood @ scene prevents the crime laboratory from having to analyze superfluous and irrelevant evidence. • Blood absorbs at 415nm … • Does not fluoresce under long-wave ultraviolet light (300-400nm – both settings on an ALS). • Tomato-based foods may or may not absorb light at 415nm (usually less so than blood) … • Give blue-white fluorescence and a yellow or yellow-orange fluorescence under long-wave UV light. Blood Ketchup • Stain on the left = bloodstain • Stain on the right is a ketchup stain. • The blood absorbs the light, which is why it appears darker. • The ketchup has a blue-white fluorescence.

24. Infrared (IR) Cameras Combining Searching and Archiving • Blood absorbs in the infrared, which makes it appear dark, • Can visualize blood on dark backgrounds that do not absorb in IR • Sometimes on dark, shiny surfaces. • In the past, • Delayed for film processing • Made on-scene usefulness problematic because it took time before the investigators knew whether the infrared light had “found” blood. • Used show blood patterns on dark surfaces where it was known to be present. • Not a mechanism for finding difficult to see bloodstains AT THE SCENE during the investigation. • Digital IR camera and the ALS are valuable on-scene partners • Tools to help locate dried blood on difficult surfaces. • Digital cameras have characteristics different than film cameras simply because they the LCD viewers allow one to “see” the blood in-situ.

25. Detecting Blood Using An IR Camera • Instantaneous peek at dark surface … does not absorb IR light.. • Camera expands an investigator’s sight range into the real-time near infrared, … IR digital camera is indispensible tool for on-scene investigations. Even 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. Legend: White arrows point to bloodstains • 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. • The stains on the carpet in photograph were invisible to the naked eye and to the settings on the ALS – the ALS could not effectively subtract out the background

26. ALS WavelengthsApplications to Finding Biological Evidence – MiniScope 400 Evidence Type ALS SettingsGoggle Camera Filter Bone 455/CSS/515 Orange Orange Teeth Fingernails Body Fluids CSS Orange 1-2 Orange Dk Surfaces UV Clear/Yellow None “ w/crust White/oblique Clear None Hair untreated Blk White/oblique Clear None treated-red/bld 415/CSS Yellow/Orange Yellow/Orange Blood 415, 455 Clear/Yellow None

27. Choosing a Goggle ColorMatches Wavelengths Color Range ALS Setting (nm) Goggle Long wave UV 300-400 Clear Violet 515-445 Yellow Blue/green 455-515 Orange Green-red 536 Red - - - - - - CSS Orange http://www.evidentcrimescene.com/cata/light/light.html

28. Detecting Blood @ the Scene

29. 1862 Chemical Presumptive Testing • Chemical tests that react with blood were developed in the mid 19th century. • Needed a method to know whether unknown stain might be blood • Chemistry narrowed the range of possible substances by approximately 95%. • Positive chemical test means that there is approximately a 95% chance that the unknown stain is or contains blood. • Many reddish or dark stains at a crime scene are not blood. • Example is stain from the spray of a shaken CokeTM can on a dark wall. • Each works on the same principle. • Two categories: Colored Dyes or Luminescence. • The former include a range of dyes that turn color in the presence of hemoglobin, a protein component of blood, and a peroxide – hydrogen peroxide is the most commonly used peroxide. • These reagents are available commercially and include a vast array of choices:

30. Common Presumptive Test Reagents phenolphthalin (Kastle-Meyer) leucomalachite green (LMG) Luminol (BlueStar) 3,3’,5,5’-Tetramethylbenzidine (TMB) leucocrystal violet (LCV) o-tolidine Benzidine: Carcinogenic o-toluidine hydrogen peroxide: Bubbles

31. Blood – Presumptive testsGeneral Considerations Step 1: Oxygen free radicals cleaved from peroxide group Heme Fe+++ Heme Fe++ 2H2O2 2O· + 2H2O Free radical interacts with organic chemicals (dyes) Step 2: Oxygen free radicals react with reduced dye O· chemical oxidized (Colored) + Chemical reduced Presumptive test detects oxidized organic dyes

32. Kastle-Meyer Test for Blood Most Common Lab & Scene Test • One step • All reagents added together • Considered to be most sensitive • Doesn’t allow for identifying false positives • Two Step • Reagent added to the stain • Peroxide added last • Three Step • Alcohol added first • Reagent second • Peroxide last Also known as the phenolphthalein test

33. Blood Reacting Chemicals that Luminesce • Other category are chemicals that react with hemoglobin and peroxide but instead of turning color, they luminesce: … • Chemiluminescence. • Luminol, BlueStarTM and fluorescein, is used primarily at crime scenes where clean-up is suspected. • Luminol and fluorescein have enjoyed a long forensic history, but BlueStar is a recently available formulation for which claims of greater and longer luminescent intensity exist. • BlueStar … two formulations … one for on-scene use … training. Since the reagent is expensive, the training formulation is a less expensive version but its manufacturers warn it will destroy DNA. • The on-scene and more expensive version supposedly does not destroy DNA. • Luminescence produced can be dramatic

34. BlueStarTM

35. The Luminol (BlueStar) Procedure • Darken room or area as adequately as possible. Sometimes covering windows, door areas, exit lights, etc, with black plastic bags will suffice. • Spray suspect area with 2% 5-sulfosalicylic acid and allow to dry. • Sulfosalicylic acid fixes the proteins in blood by denaturing them • Set camera on tripod, set aperture to “bulb,” turn off lights and take photograph of scene using a 2 minute exposure. Check that photo is not over exposed. If over exposed, adjust shutter speed. • If exposure is adequate (see scene detail in photograph), trip shutter and spray area with BlueStarTM. Allow luminescence to develop. • When fluorescence begins to fade, spray the area again. Continue this process for the entire two minutes. • Then trip the shutter and observe the photograph.

36. Hemascein Hemascein®, a non-luminol formulation, designed to Qualitatively reveallatent bloodstains at a crime scene.

37. How Hemascein Works • Hemascein® detects latent bloodstains • using a novel fluorescein as active component • Area suspected of containing latent bloodstains sprayed with Hemascein® using ABASpray™. • Hemascein® reagent is reduced by hydrogen peroxide (colorless) and then oxidizes fluorescein. • Fluorescence comes after excitation an ALS • Use ALS between 415 and 480 nm. • After locating suspect blood, test for human origin using immunochromatographic test. • Evidence swabbed and transported for DNA analysis. http://www.abacusdiagnostics.com/howitworks.htm

38. So Many Choices: What to Use When? • The choice of which to use and when is important. • Scene where an informant says that individual had been murdered years earlier. • Finding the blood visually or even with an ALS might be fruitless. • Maybe the original scene had been remodeled or repainted. • The team leader needs 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 spray using BlueStarTM ? • Can the team darken the room sufficiently? • OR Should use a reagent that forms a color, such as leuco crystal violet OR KM. • Suppose investigation based on informant’s information that the room had, been painted to hide blood, • Consider possibility of finding the blood “under” the paint and discuss how to accomplish that. • One consideration is spraying with BlueStarTM.

39. Confirming Human Bood

40. Lateral Flow Immunochromatography Rapid technique for identifying small amounts of specific molecules. • Forensic application largely used to identify blood, semen, saliva and urine. • Can be conducted on-scene, BUT reagents must be purchased commercially, which raises the per/test cost significantly. • Not a presumptive test: • Specifically identify unknown stains as human blood • Some cards cross react with ferret blood, • Semen or saliva (identify salivary amylase). • Tests purchased as testing kits, • Must pass quality tests for sensitivity and specificity. “Immunochromatography strip test, or namely lateral flow test, is a simple device intended to detect the presence or absence of the target analyte.” It’s a form of immunoassay. Well-known examples are in-home pregnancy tests. http://www.creative-diagnostics.com/Colloidal-Gold-Lateral-Flow-Strips-Development.html?gclid=CMCC8-ry9LUCFY6e4Aod1lsA1Q

41. Immunochromatographic Cards • Problem with immunochromatographic cards, especially those from Abacus and OTEB is that they suffer from what is known as the Hook Effect. • Happens when testing overly concentrated samples of human blood • False negative test, • Incorrect and potentially misleading result obtained. • RSID cards do not demonstrate a Hook Effect • When the Hook Effect occurs, sample must be diluted and re-run. • Quickness, ease-of-operation, specificity and sensitivity of these immunochromatographic cards makes it tempting to avoid the traditional chemical tests entirely. • If cost is not an issue, this might be a best choice because these tests confirm the presence of human blood in a single test. • Used immune-card and/or stain extract can be submitted to the laboratory for DNA analysis, • Should save the laboratory time in selecting certain scene stains from submitted crime scene samples for DNA analysis, • Laboratories normally prefer to extract the samples

42. Immunochromatographic Cards • Employing immunochromatographic cards exclusively • Mistake, if all blood tested turns out to be nonhuman. • Negative test typically means that human blood is not present. • False negatives occur with highly concentrated blood extracts … because of the Hook Effect. • Exception is the blood test by RSID which has no Hook effect. • True negative occurs when there is insufficient human blood present; all tests have limits of sensitivity. • Of the immuno-cards available, the Abacus Diagnostics card for blood is the more sensitive; • HemaTraceTM card detects lower amounts of blood than the RSID card. • Investigators • Need to be aware of nuances among products • Should test cards … for sensitivity and specificity as part of the validation of a comprehensive quality assurance program. Used with Permission from Dr. Reena Roy Penn State University

43. Collecting Biological Evidence for DNA Analysis

44. Touch DNA Common Buzzword in Modern Investigative Forensics

45. Touch DNA • Cellular material is biological evidence with DNA. • Perpetrator holds a weapon or picks up an object with an ungloved hand, cellular material transfers from the hand to the object. • Much modern forensic DNA analysis involves what has become known as “touch evidence.” • All biological evidence must be considered from the perspective of its location at the scene, its pattern as well as its donor.

46. Touch DNA vs Low Copy Number (LCN) DNA • Touch DNA is not Low Copy Number (LCN) DNA. LCN DNA profiling allows a very small amount of DNA to be analyzed, from as little as 5 to 20 cells. • Touch DNA testing involves analyzing “normal” amounts of DNA • Humans shed tens of thousands of skin cells each day, and these cells are transferred to every surface our skin contacts. When a crime is committed, if the perpetrator deposits a sufficient number of skin cells on an item at the scene, and that item is collected as possible evidence, touch DNA analysis may be able to link the perpetrator to the crime scene. • Touch DNA has been successfully sampled from countless items including gun grips, steering wheels, eating utensils, and luggage handles, just to name a few. • However, since Touch DNA is usually deposited in smaller amounts than the DNA found in bloodstains or other body fluids, it is more difficult to obtain DNA profiles from touch DNA samples. • The key to obtaining successful Touch DNA results depends on recognizing items which may be suitable for Touch DNA analysis and using the sampling technique that will recover the highest number of skin cells.

47. Touch DNA • Potential Evidentiary Value • Consider the potential evidentiary value of the DNA. • Account for the relationship between the victim and the suspect (if one exists), • Consider any possibility of “innocent transfer” of DNA that may have occurred before the alleged crime. • If suspect is a family member, and either lived with, or had recent contact with the victim, • Finding suspect’s DNA on the evidence may be of limited probative value. • Touch DNA can easily be transferred throughout the household via day-to-day interactions, contact with furniture items/bedding, or through the laundry.

48. Collection Methods Thinking Though Where The DNA Is • “Swabbing method”, • Surface of item is rubbed with a cotton swab to collect possible cells. • This method preferred for hard items such as glass or plastic. • “Cutting method” • Used for soft items, such as clothing, in which fabric from areas of interest is cut to collect possible cells. • “Scraping” and “Tape Lift” methods • Surface of soft items (such as clothing) are either scraped with a blade, or sampled with a small piece of tape, to collect possible cells. • Larger surface area can be sampled. • An increase in surface area increases the number of possible cells recovered; therefore, increasing the chances of obtaining a DNA profile. • Ideal in situations where the scientist can locate areas on the item which are most likely to contain the perpetrator’s skin cells. • Clothing left at scene by the perpetrator • Pressure points on the clothing such as the interior neck of a shirt or the band inside a hat, are excellent candidates for these sampling methods. • Sexual assault case where victim’s clothing removed by the perpetrator, areas such as the waistband may contain sufficient cells belonging to the perpetrator to produce a profile.

49. Thinking Through Evidence Collection • Sexual assault by a stranger, finding the suspect’s DNA anywhere on the victim’s clothing may have evidentiary value. • Gather as much information from victim as possible (if living), • Attempt to recreate the events if the victim is deceased. • If the victim’s pants pulled down, then the investigator and forensic scientist should consider sampling areas for Touch DNA where one would envision that the suspect would have grabbed during the assault. • Finding the suspect’s DNA on the victim’s clothing, and in certain areas of the clothing, may help corroborate the victim’s version of events and help address the allegations in ques­tion. • Attempt to collect clothing of deceased individuals • Collect samples from the clothing prior to the deceased being removed from the scene. • Collecting clothing at the scene PLUS optimal preservation allows obtaining Touch DNA at a later date, even if it’s not initially indicated to be present at the crime scene. Channeling Information • Provide lab scientist with case background information in order to receive the best advice on the potential value of DNA evidence • Crime scene photos can be quite useful. • Lab scientist should have appropriate questions/suggestions for the investigator to answer or consider.

50. Limitations of Touch DNA • Touch DNA sampling methods, and DNA processing procedures are very sensitive. • Detecting contamination from law enforcement personnel or sampling investigator • Even when appropriate PPE is worn. • May be necessary to obtain elimination samples from key personnel in the case where foreign DNA profiles are obtained that cannot be attributed to a suspect or the victim. • Also an increased chance of obtaining mixed DNA profiles containing DNA from individuals that may have come into contact with the victim/evidence item near the time of the crime. • Contributors to these mixtures could include the victim’s spouse or children • What does unexplained DNA mean? • Foreign male profile from a Touch DNA sample may be obtained from evidence pertaining to a female victim. • If the male DNA profile doesn’t match the suspect in question the investigator needs to consider its relevance to the case. • The foreign profile could from the true perpetra­tor and the original suspect could be innocent. • DNA profile could be from adventitious transfer from crime scene personnel, first responders, laboratory analysts, or crime scene equipment such as fingerprint brushes. • Need to evaluate and address these questions before moving forward with the investigation. • Some evidence items are also not recommended for the collection. • Severely degraded DNA - moldy clothing • Samples exposed to extreme environmental conditions • Weapons left outside for months or years, have been washed, or are heavily soaked in the victim’s body fluids. • Items likely touched by many people: public pay phone or store counter.