Biological Evidence and Serology PDF

Module 1: Crime Scene Investigation Biological Evidence Page 1 Module Overview Introduction Forensic science is the application of science in a legal environment. An operational forensic laboratory receives a wide range of cases from minor burglaries to more serious cases such as sexual assault or m...

Module 1: Crime Scene Investigation Biological Evidence Page 1 Module Overview Introduction Forensic science is the application of science in a legal environment. An operational forensic laboratory receives a wide range of cases from minor burglaries to more serious cases such as sexual assault or murder. Therefore, the evidence submitted to the forensic laboratory and the tests conducted by a forensic scientist can vary substantially from case to case. Irrespective of the case, the aim of the forensic scientist should remain the same. That is to objectively examine the evidence in a scientific manner and present the results clearly. First we need to define the role of the Forensic Serologist in cases involving biological evidence and to provide an overview of the types of evidence routinely analyzed and the basic methods of obtaining evidence from the crime scene. Objectives At the completion of this module students should: Understand the basic role of the forensic serologist in analyzing biological evidence Be familiar with the types of samples routinely analyzed by the forensic serologist Be familiar with the general processes of crime scene processing Have a general understanding of crime scene investigation in relation to obtaining biological evidence Describe and record the methods of location and collection of biological evidence Understand the measures needed to prevent contamination of evidence Understand the measures needed for protection from biohazardous samples and waste Module 1: Crime Scene Investigation Biological Evidence Page 2 What is Forensic Science? What is Forensic Science? Forensic Science can be defined as the application of science as it pertains to the law. The basic tenet of forensic science is based on the observations by French doctor and forensic pioneer, Edmond Locard (1877-1966), who recognized that when two objects came into contact that there was a bi- directional transfer of material between the two objects. His observations became known as the Locard Exchange Principle; widely quoted as “Every contact leaves a trace”. Over the last century, the field of Forensic Science has evolved into many distinct specialties with disciplines that include Toxicology, Drug Chemistry, Biology/Serology, Trace Evidence, and DNA typing. Related fields that may be performed by enlisted officers as opposed to Forensic Scientists include Ballistics, Blood Stain Pattern Analysis, and Crime Scene Investigation. Modern Forensic Science also now includes niche fields such as Meterology, Engineering, Electronics, and Virology. Once its own forensic discipline, Forensic Serology is now sometimes referred to, by some agencies, as Forensic Biology since the development of DNA testing superseded the traditional serological techniques of personal identification. Adopting ASCLD-LAB terminology many labs are again renaming their Forensic Biology sections as Forensic Serology. For this reason and for the purposes of this course, we will use the terms Forensic Serology and Forensic Serologist. Forensic Serology is best described as the investigation and analysis of biological evidence, such as blood and other bodily fluids and tissue, as a means of identification and reconstruction of crime scenes. In 1901 Karl Landsteiner first observed the agglutination of human blood cells with human sera and determined that blood could be distinguished by a specific group or type, leading to the development of the classic ABO blood grouping system we still use today. From identification using blood grouping the discipline has further evolved into the more discriminating application of forensic DNA testing. Historical Overview of Forensic Serology 1800s First attempts were made to identify reddish brown stains on clothing as blood, using microscopy and chemical tests for the presence of hemoglobin; these were the first presumptive tests developed for blood identification 1853 The first confirmatory test for hemoglobin was developed Ludwig Teichman (Poland) 1901 A method for distinguishing between animal and human protein was first published in the scientific literature Paul Theodor Uhlenhuth 1901 Differences were identified between human blood types that formed the basis for ABO blood types; agglutinins were identified Karl Landsteiner (Austria) 1915 Methods were developed for grouping dried blood stains Leones Lattes ( Italy) Rhesus blood factors were identified Landsteiner and Weiner 1949 The Ouchterlony species identification test was developed A. Ouchterlony (Sweden) 1960 Tests were developed for species identification using mixed agglutination methods Stuart S. Kind (UK) 1964 Polymorphic variants of the phosphoglucomutase (PGM) Brain Culliford (UK) (Germany) enzyme system were identified 1970s Further research determines that polymorphic variants exist for a number of serum proteins and blood enzymes 1984 Seminal work in DNA fingerprinting was first published in the scientific literature 1986 DNA profiling applied to the forensic case and the identification of Colin Pitchfork Alex Jefferies 1986 The polymerase chain reaction (PCR) techniques was invented 1988 FBI begins using DNA analysis in casework 1992 The National Research Council publishes its first report (NRC I) DNA Technology in Forensic Science 1993 The first STR it becomes available 1996 The National Research Council publishes its second report (NRC II) The Evaluation of DNA Evidence 1998 FBI establishes CODIS DNA database 1998 DNA Advisory Board (DAB) establishes quality assurance standards for forensic DNA laboratories. Kary Banks Mullis Module 1: Crime Scene Investigation Biological Evidence Page 3 Crime Scene Investigation Crime Scene Investigation Blood and other biological fluids may be recovered as evidence in violent crimes. This evidence is commonly found as blood from the victim on the clothing of an assailant, as semen from an assailant recovered from a rape victim, or perhaps as a series of blood spatters left at a crime scene. Less obvious forms of evidence include body fluids such as saliva, urine, vomit, and amniotic fluid; body parts, human remains, and tissues associated with mass disasters and homicides, as well as injuries inflicted in vehicular hit and run accidents. Although such samples contain markers of individuality, the individualizing tests may be hampered by sample degradation; the occurrence of mixed fluids; poor sample integrity, and the challenges of analyzing samples mixed with chemicals, textiles and dirt. It is the responsibility of the forensic serologist to determine which biological stains are probative to the investigation. The forensic serologist is often instrumental in triaging evidence as it journeys from a crime scene to identification via DNA typing. They may characterize recovered biological fluids such as semen, saliva, blood and urine, perform both presumptive and confirmatory tests on these fluids, and present their findings as an expert witness in a court of law. Principles of Crime Scene Investigation The forensic serologist can encounter a vast array of physical and biological evidence in the laboratory and in some cases may be called to help or guide the collection and interpretation of evidence at a crime scene. Successful and meaningful evidence analysis in the laboratory depends on a thorough investigation of the crime scene and the appropriate collection and preservation of the biological evidence. Some of the most important aspects of crime scene investigation include: securing the scene the prevention of contamination the preservation of physical evidence maintaining chain of custody through proper documentation Module 1: Crime Scene Investigation Biological Evidence Page 4 Securing the Scene Securing the Scene Personnel responding to a crime scene must enter the scene with caution and be cognizant of other personnel, vehicles, ongoing events, potential evidence, and environmental conditions. Initial observations such as the presence of distinctive odors or sounds may be of use at a later time in the investigation. In some circumstances there may be a secondary crime scene or multiple crime scenes to investigate and process. On arrival at the scene the forensic investigator should assess the site and consider any safety measures that need to be taken to protect themselves and other personnel. Securing the crime scene area is the responsibility of the initial responding officer (IRO). The IRO identifies anyone present at the scene, and takes appropriate steps to prevent the alteration or destruction of physical evidence by restricting movement and scene activity. Establishing, defining, and controlling the boundaries of the scene facilitates the maximum preservation of evidence. These boundaries are determined by the location of the scene and the type of crime committed. As a general rule, boundaries start at the center of the scene and extend outward to include: Where the crime occurred Potential points of entry and exit of suspects and witnesses Places where the victim or evidence may have been moved Boundaries are critical for the control of evidence integrity and can be created by setting up physical barriers, by controlling the flow of personnel into and out of the scene, documenting the entry of persons entering or leaving the scene, and preventing the loss, or compromise of evidence from the effects of weather, foot traffic and vehicular damage. Processing the Scene The first step in any routine crime scene investigation is to establish a plan of action and a crime scene reporting protocol. A basic protocol for scene processing is summarized below: Surveying the scene Photo/Video documentation and searching the scene Mapping and measuring the scene Recording and documenting the location of physical evidence at the crime scene Searching for fingerprints and other physical evidence A forensic serologist may be asked to assist in processing the scene, especially if there is a need to decide which evidence may be probative in a complicated scene. The serologist may be asked to utilize specialized testing techniques such as alternate light sources and blood visualizing chemicals such as Luminol or Blue Star. Each person involved in processing a crime scene must do so in an organized and routine manner. Every effort must be made to properly preserve and document biological evidence. Care must be taken to minimize the loss of trace evidence such as hair and fibers, and proper collection, packaging and storage is tantamount to optimizing possible DNA results. Since biological evidence is prone to degradation, it is important for officers collecting it to follow protocols set in place by the forensic laboratory. Recording the Scene All activities and observations made at a scene, must be documented to preserve the events and information. Documentation may take the form of sketches, written notes, photography and video. Documentation ensures the integrity of the investigation and provides a permanent record of the scene for later evaluation. Records include observations such as the location of persons, vehicles, or items within the scene, and the condition and appearance of the scene upon arrival. Most records are made by the investigator in charge, and scene processing personnel during the scene walk -through. A walkway other than the established path of entry should be used to reduce contamination. Information can be recorded on preprinted documents or forms designed for recording different aspects of crime scene investigation. Many of these documents, which may also be used in the laboratory as part of the note taking and searching part of evidence analysis, have been developed and refined from years of actual case experiences. The design of the document may vary, but the purpose and goals behind their use are identical from one agency to another. These templates may include body outlines for recording injuries, clothing outlines for recording damage or staining, and data analyses sheets for the simple recording, reference to, and interpretation of, basic laboratory and presumptive tests. Module 1: Crime Scene Investigation - Biological Evidence Page 5 Identification, Collection, and Preservation of Evidence Identification, Collection, and Preservation of Evidence During the walk -through, fragile and perishable evidence should be identified and protected. This evidence should then be documented, photographed and collected. During processing of a crime scene, contamination is controlled by limiting access to those directly involved in scene processing. Specific areas should be designated for trash and equipment, and single-use materials should be used for the collection of biological materials and other trace evidence, to minimize cross contamination. The collection of evidence should be prioritized to minimize loss and contamination. At a crime scene a secure area is generally identified for the temporary storage of evidence in accordance with rules of evidence, and a chain of custody is established. A systematic search pattern, usually determined by the size and nature of the scene, is used for the location and collection of evidence. Several different search patterns may be used depending on the nature of the crime scene being processed: Strip or lane search Grid search Spiral search Quadrant search As an example, spiral searches are generally used in underwater crime scenes. The search will begin where the key evidence (such as a body) is located and radiate and extend outwards in a spiral pattern. The way physical evidence is handled is one of the most important factors of an investigation. The collection of evidence must be documented, and should include records of its location at the scene, the date of collection, and who collected it, as part of the chain of custody. During evidence collection, the investigator may also collect reference materials such as fibers from carpeting, footwear, or tires for comparison purposes. Once the scene has been processed, a final walkthrough is conducted to ensure that all evidence has been collected and that materials are not inadvertently left behind. Resources Go to http://www.crime -scene-investigator.net/collect.html (http://www.crime-scene- investigator.net/collect.html) for an overview of the evidence collection guidelines for the following types of evidence that are routinely recovered in serology/biology forensic cases: Bloodstains Semen stains Hair Fibers Module 1: Crime Scene Investigation Human Remains and Identification - Biological Evidence Page 6 Human Remains and Identification In a homicide investigation, the forensic investigator has a responsibility to help establish the nature of death; whether it is accidental, homicide or suicide, while preserving any evidence that's present at the scene. Moving the body, clothing, or any other items covering a corpse may result in the loss of evidence. Notes taken at this type of scene should include the following details: Position of the body Description and state of clothing (is it partially removed, stained, tangled or twisted?) Describe the location, intensity, size, direction of flow (if possible) of any staining Type of stain (blood-like, semen, dirt, vomit, feces, urine or some other fluid Any visible damage to the clothing and body Description of blood staining (spots, spatter, or smearing and directionality) Appearance of evidence removal (has the body been washed or blood cleaned from the body?) Estimated amounts of blood (has staining penetrated layers, carpeting or other surfaces, is it superficial?) A corpse that is located outdoors, or that has apparently been moved from another location, warrants some special consideration since the remains may be partially decomposed or damaged by insects or animals, or crucial evidence may be lost at the original location. As before, the careful examination and application of standard procedures for maximum crime scene and evidence protection should be applied to the collection of evidence that includes trace evidence and body fluids. The recovery and analysis of human remains is a complex process since the body may have been subject to a large degree of decomposition and alteration. Some skeletal parts and some organs may remain, which together with clothing fragments, may provide useful evidence. Human remains are found in a variety of places including outdoors, but also, in attics, furnaces, and basements. A body may have been buried in soil, frozen, covered with brush or overgrown vegetation. With the recovery of outdoor remains, clothing and evidence may be spread over a wide area and may have been subjected to animal interference and insect infestation. With appropriate care and procedures, all of these scenarios can still provide large amounts of evidence. Estimating Time of Death In murder, accident, and suicide cases, the time of death is an important detail for the investigation. Using post mortem changes in the body, an approximate time can often be determined. The following parameters are usually given consideration: Changes in the eyes such as dulling of the cornea and the appearance of a film Body temperature cooling that can be related to the outdoor conditions The temperature of the body at the time of death Whether the body is clothed or unclothed The presence of rigor mortis The presence of post mortem lividity Degree of decomposition Algor Mortis is the cooling of the body after death when the ambient temperature is cooler than the body temperature. A nearly nude body will generally cool at a rate of 1.5 0C per hour, for the first 8 hours, when the ambient temp is 18-200C. Many factors can cause a body to deviate from 370C plus environmental conditions such as heat cold, burial etc all affect cooling rate too. A body usually reaches ambient temperature 18 to 20 hours after death. Rigor Mortis is rigidity that appears 6 to 12 hours after death due to biochemical changes in the muscle tissue. The degree and extent of rigidity can be used to estimate time of death. Rigidity remains for 2 to 3 days then disappears again. When blood circulation stops, blood pools in the lower extremities of the body due to the effects of gravity, causing a purplish red appearance called Livor Mortis. Lividity appears 1 hour after death and is fully developed within 3 to 4 hours. The location of lividity on the body can also be used to determine whether a body has been moved from its original position. Putrefaction is the breakdown of the body due to autolysis and bacterial decomposition. These events convert tissues of the body into liquids and gas that cause discoloration, bloating, and swelling. Within a short period of time, fluid filled blisters appear on the skin, and the pressure of internal gases can force stomach contents out of the mouth, nose and anus. Although submersion in water results in similar effects, the processes are generally slowed due to colder temperatures. Microorganisms present in soil also decompose buried bodies. Within one to 3 years, all soft tissues will have disappeared leaving only bones. Bodies may be preserved by mummification or through the formation of adipocere, a waxy covering which begins to develop in 6 to 8 weeks as body fats are hydrolyzed. Module 1: Crime Scene Investigation Biological Evidence Page 7 Crime Scene Analysis Crime Scene Analysis Use of Crime Scene Photography Crime scene photographs are permanent pieces of evidence that may be presented in a court of law to prove or disprove facts or issues in question. Although an investigator can verbally describe a crime scene, photographs can be used to present the same facts in a more accurate and easily understood way. Photographs can be useful to document blood stain patterns, footwear impressions and injuries to both victims and suspects. Bloodstain Interpretation Blood evidence can provide information that may be used to solve a case, so correct documentation, collection, and preservation of blood evidence is very important since improper handing can destroy its evidentiary value. Properly collected and preserved blood evidence may be used to link an individual to a criminal act or a crime scene; to strengthen or contradict a witness or suspect’s statement, or to eliminate a person as a potential criminal suspect. Properly documented, collected, and stored blood evidence can be presented in a court of law several years from the time of the criminal act. Bloodstain Pattern Analysis The shape, appearance and distribution of blood drops at a crime scene may be used to reconstruct a crime. The shape and appearance of bloodstains and smears on clothing or other surfaces may also provide useful information about a crime. Vehicles as Crime Scenes A vehicle used for a murder or to transport a victim may hold an array of physical evidence. In some cases the victim may still be present in the vehicle, or the victim may have been removed. The procedures for searching in these scenarios are the same as those at any other crime scene. Care should be taken with the vehicle interior as limited space for working may cause physical and trace evidence to be lost or destroyed. Hit and Run Investigations In hit and run cases there are two types of scenes to process: Damage to other vehicles or property Death and injury Physical evidence can exist which may Identify the missing vehicle Connect the vehicle with the crime scene or the victim Help in reconstructing the events surrounding the crime Types of evidence that may associate a vehicle with a scene or victim include: 1. Hairs 2. Clothing fibers 3. Paint 4. Glass 5. Blood 6. Tissue Module 1: Crime Scene Investigation Biological Evidence Page 8 Crime Scene Safety Crime Scene Safety When processing a scene, or conducting a laboratory investigation, an investigator may face a number of potentially hazardous situations. Investigators routinely handle potentially biohazardous materials and chemical agents through the collection, processing, and examination of evidence. Although the investigator is ultimately responsible for recognizing chemical, biological, and physical hazards, it is the responsibility of their organization to develop policies and programs on health and safety practices; to provide training on the application of these principles, and to ensure these practices are followed. In this course we will mainly consider exposure to blood borne pathogens. However, some of the analytical techniques employed by the serologist may result in or risk exposure to toxic chemicals. Routes of Exposure The most common hazard faced by the forensic serologist is exposure to potentially infectious human blood and body fluids. There are several routes of exposure to biological and chemical hazards to be aware of: Inhalation of airborne pathogens (anthrax) and volatile chemicals Skin Contact - a frequent route of entry into the body for chemicals and biological hazards. Chemicals can cause local irritation, redness, swelling, burning or damage to the contact tissues. Severity of the reaction depends on the type and concentration of the chemical, and duration of exposure. Systemic effects can be as minor as dizziness and nausea, and as extreme as organ damage, shock, and unconsciousness. Ingestion - Ingested corrosive materials can damage the mouth, throat, and digestive tract. Ingested toxic chemicals are usually absorbed by the body via the stomach and intestines. Regular hand washing, and abolishing the consumption or presence of food or drink in contamination prone areas, will minimize exposure to chemicals or biological contaminants by this route. Injection - Needle sticks and mechanical injuries from contaminated glass, metal, or other sharp objects can introduce biological contaminants directly into the bloodstream. Biological Hazards A forensic investigator routinely comes into contact with body fluids, tissues, and biological specimens that may be contaminated with blood borne pathogens. Pathogens passed on from biohazardous materials and waste can cause life threatening and debilitating diseases such as HIV, AIDS, and hepatitis (inflammation of the liver). Blood Borne Pathogen Safety In 1991, the Occupational Safety and Health Administration (OSHA) issued Title 29, part 1910.1030 of the Code of Federal Regulations (CFR), Occupational Exposure to Blood borne Pathogens (BBP). Occupations considered being at risk included including law enforcement, emergency response, and forensic laboratory personnel. Universal Precautions Universal precautions are the fundamental mechanisms considered necessary to maintain the BBP standard and are the primary mechanisms for infection control. The BBP standard directs employees to treat all human blood, body fluids, or other potentially infectious materials as if infected with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). A number of protective measures should be taken to avoid direct contact with these potentially infectious materials: Barrier protection - disposable gloves, coveralls, and shoe covers must be worn when handling potentially infectious materials. Appropriate eye and face protection - worn to protect against splashes, sprays, and spatters of infectious materials. Similar precautions may be followed when collecting dried bloodstains. Place contaminated sharps in closable, leak proof, puncture-resistant containers when transported or discarded. Label the containers with a Biohazard warning label. Prohibit eating, drinking, or applying cosmetics where human blood, body fluids, or other potentially infectious materials are present. Well-fitting respirators can be worn when there is potential for contamination or transfer from airborne biohazards. Module 2: Processing Biological Evidence Module Overview Page 1 Introduction Nuclear DNA is found in human skin cells, skin tags on hair roots, and in the heads of sperm cells. It is not found in red blood cells (RBC). The DNA extracted from human cells and tissue has been demonstrated to be suitable for nuclear DNA typing. Despite these readily available sources of DNA, samples are extremely susceptible to damage, degradation and contamination. Improper handling, collection, packaging and storage can easily compromise the integrity of a sample and care must be taken at times and during all steps of processing to insure optimum results. This module will address the types of biological samples encountered in forensic serology/biology and DNA Testing. Objectives At the completion of this module students should: Be familiar with the types of samples routinely analyzed by the forensic serologist Be familiar with the general processes of crime scene processing Have a general understanding of crime scene investigation in relation to obtaining biological evidence Be able to describe and record the methods of location, collection, and storage of biological evidence Module 2: Processing Biological Evidence Page 2 Searching a Crime Scene for Blood and Other Body Fluids Searching a Crime Scene for Blood and Other Body Fluids Forensic Serology is the detection, identification, and typing of bodily fluids as it pertains to criminal investigations. The ability to critically observe the scene and accurately record those observations while maintaining the integrity of evidence are characteristics of a good crime scene investigator. The investigator often works in conjunction with the forensic serologist in selecting which biological evidence is anticipated to be most probative in understanding a crime. Communication between the two is very important in the triage of evidence. Back in the laboratory, the forensic serologist must also meticulously observe physical evidence, conduct tests, and record findings. Following analysis of the evidence, the forensic serologist must be sure to repackage it securely and store it properly so as to maintain it's integrity and chain of custody. Biological evidence is most informative in cases when the suspect and victim come into close contact or struggle with each other. In stabbing and beating incidents, the close proximity of the assailant to the victim can result in the transfer of blood between the two. During assailant-victim struggles, skin and blood may be transferred to the victim’s fingernails. Throughout this section we consistently refer to the processes of locating and processing of bloodstains, however the principles addressed are equally applicable to locating, collecting, and preserving all types of biological fluids and stains. When processing a crime scene, the most fragile evidence is collected first, and then more obvious evidentiary items are identified next. In some case scenarios, special techniques may be used to locate some of the "hidden" evidence. It is not unusual for a perpetrator to try to conceal or hide evidence by cleaning up blood-contaminated areas. However, blood is actually very difficult to remove from a scene completely. Since blood is a fluid, it can flow between floorboards, under baseboards, and into carpeting. In such cases, it may be more useful to conduct searches using high intensity light sources that can aid in the visualization of bloodstains and other body fluids, thereby minimizing the loss of evidence at the scene. Module 2: Processing Biological Evidence Page 3 Searching Techniques Alternate Light Sources Since the use of visible light may be insufficient to reveal or locate some types of biological evidence, other types of light may be necessary. Alternative light sources (ALS) may be used to reveal hidden or undetected biological fluids and evidence. ALS systems may be a series of light sources that resemble flashlights, each emitting a different wavelength of light. The analyst wears goggles with filters which coordinate to each wavelength of light. These filters both protect the analysts eyes from ultraviolet light and also enhance visualization of different biological fluids. An example of available wavelengths of light and uses follows: White light 400-700nm (blood spatter and bite marks) UV light 350-380nm (bite marks, body fluids, fibers) Violet light 400-430nm (blood spatter, bite marks, body fluids, hair, fibers) Blue light 420-470nm (blood spatter, bite marks, body fluids, hair, fibers, gunshot residue) Blue/Green light 445-510nm (bone, teeth, fibers, gunshot residues) Green light 480-560nm (bone, teeth, fibers) Orange light 570-610nm (fibers) Red light 610-660nm (fibers) Infrared light 800-900nm (blood spatter, fiber, gunshot residue) As you can see, biological fluids may be observed by several different wavelengths of light. An analyst may scan for evidence using several wavelengths as well as several different filter colored goggles. Under UV light, blood doesn’t fluoresce but shows up as a black/dark area, which can be prominent if the rest of the item fluoresces. The detection of blood in obscure areas can often be carried out with a high intensity light, enabling samples to be located, obtained and taken to the laboratory for further testing. Light sources can also be used to provide oblique (side) lighting, which is an excellent means of finding trace evidence and other small items of interest especially on large items such as bedding, comforters and carpets. In, the laboratory setting, the ALS is very useful in searching items for the exact location of blood, semen, saliva, and urine. Frequently, light in the range of 400-510nm causes fluorescence of stains such as semen, saliva, and urine using orange or yellow filters. By narrowing down the exact location of a biological fluid, a forensic serologist may better focus testing and ultimately a DNA profile. Luminol In some scenarios, especially if a crime scene is suspected to have been cleaned up, chemical enhancement might be used to locate or identify areas of suspected blood staining. When Luminol is applied to bloodstains the stains appear to glow in the dark due to chemiluminescence. Luminol is utilized as a last resort for locating stains that may be very dilute or at times to help visualize spatter patterns. Although luminol is one of the most sensitive chemical enhancements available, it does have drawbacks. It is not specific, at times reacting with metal, cleaning products containing bleach, and even things like paint. Additionally, Luminol is a respiratory irritant, so care must be take to ensure proper ventilation. For genetic analysis to be carried out on a sample, enough stain must be recovered to allow confirmatory tests to be conducted. This amount of blood is usually sufficient when visible to the naked eye. If a stain is not visible without Luminol treatment there may be insufficient sample for further confirmatory or genetic testing, especially when the stain is diluted from being sprayed with a liquid enhancer. Complete darkness is also necessary to view the luminescence created by a positive Luminol reaction, which can be difficult to achieve at some crime scenes. The requirement for darkness can hamper processing and photography of any potential evidence revealed. Leucocrystal Violet (LCV) LCV is a reagent that reacts with heme in blood to produce a violet color. Like Luminol it can be used to identify latent blood prints and to enhance visible blood stain patterns. This reagent is usually used on porous surfaces and has been successfully used to enhance bloody footwear impressions and bloody foot and fingerprints. Amido Black Amido black is a highly sensitive stain that produces a light to medium blue color when it reacts with blood proteins. Results with amido black can be difficult to discern on multi colored surfaces and porous surfaces, but it works very well on non-porous surfaces to enhance latent fingerprints and other impressions in blood. One drawback to using Amido black is that it requires a rinse step which could dilute genetic material. Module 2: Processing Biological Evidence Page 4 Collection and Preservation of Biological Evidence Collection and Preservation of Biological Evidence During the evidence collection phase of a crime scene investigation, the events that occurred at the scene are not always immediately obvious. Therefore it is prudent for the scene examiner to collect and document evidence in a way that the integrity of the evidence is preserved in case further examination of the evidence is required at a later date. Crime scene investigators also need to determine what evidence needs to be forwarded to specialty sections of the laboratory for detailed analysis (serology, trace evidence, ballistics, fingerprinting). Because evidence may need to be analyzed by several different units, communication between analysts and submitting law enforcement agencies is very important. Testing may need to be prioritized to maximize probative results. Analysis of biological evidence requires the comparison of the evidence with standard reference samples obtained from the victim and suspect. Packaging Most items of evidence will be collected and packaged in clean, unused paper containers such as packets, envelopes, and bags. As a rule of thumb never package in plastic. However as we discuss this further we'll address some situations where this rule doesn't necessarily apply for certain types of evidence. Frequently, the investigator will wrap each item of evidence separately in clean white butcher paper to minimize the loss of trace evidence. The wrapped evidence is then placed in a paper package. Each package should be sealed with tamper-proof tape (never stapled), the seal initialed, and the package labeled with such information as case number, item number, and location of collection. In the laboratory the analyst receiving the evidence will check to make sure this seal is still intact and hasn’t been tampered with. Careful and complete documentation is essential in maintaining the chain of custody, a document which details who has control of the evidence and in what location at all times. Module 2: Processing Biological Evidence Page 5 Packaging of Stained Items Packaging of Dried Stains If possible, stained items should be should be packed in a paper bag or envelope and transported whole to the laboratory. By having access to the entire item the analyst will be able to decide which areas of the staining should be further tested, while decreasing the risk of contamination or stain dilution by using other collection methods. Bulkier items such as vehicles, furniture, or flooring are more difficult to handle and cannot be transported whole. If the stained item is extremely large or not easily transported, samples can be removed from the item in a variety of ways. These methods include: cutting out a manageable section, removing stains using moistened cotton swabs, scraping off dried staining, and utilizing tape lifts to remove staining. It's important to remember that if a moistened swab is used to collect a stain, the swab needs to be fully air dried before it is packaged in a paper packet. Packaging of Wet Stains Wet stains, especially those from hard surfaces, are typically collected using cotton tipped swabs. Although an effective means of collection for wet stains, when this technique is used to collect dried stains the cotton must be moistened which may result in stain dilution. Care should be taken to use the smallest amount of water possible to moisten the swab and also to fully dry the swab before packaging. Although the general rule is no packaging in plastic, large moist or wet biological evidence may be collected in clean, unused plastic containers at the scene to prevent contamination of other evidence prior to transportation. However, the storage time in sealed plastic must be absolutely minimal (less than two hours) to avoid bacterial degradation and sample putrefaction, which can destroy or alter evidence. Many agencies have drying cabinets or areas where wet evidence can be hung to dry. The evidence should be packaged in paper once it is completely dried. Care must be taken to minimize the loss of trace evidence. This can be done by placing a clean piece of white butcher paper under the evidence as it dries. Teeth and bone fragments and stomach contents are samples that can be safely stored in plastic containers before processing further. DRYING CABINETS Items that may cross contaminate each other must always be packaged separately, and containers should be closed and secured to prevent the mixture of evidence during transportation. To establish and maintain chain of custody, each container should have the collecting person's initials; the date and time the sample was collected; a complete description of the evidence and where it was found; and the investigating agency's name and case number. Module 2: Processing Biological Evidence Sexual Assault Page 6 Sexual Assault In instances of sexual assault, evidence is most frequently collected by a sexual assault nurse examiner (SANE) in a hospital setting. It's important for the victim to report for an examination as soon as possible, especially before showering since biological fluids can be lost rather easily. Typically, semen does not persist in the vagina past (5) days, so time is of the essence. SANE use kits containing pre-labeled paper envelopes. Using moistened swabs, they collect samples from areas such as the victim's mouth, vagina, anus, and any areas of possible bodily fluids (i.e. bite marks, oral contact, areas potentially containing semen). The kit usually also contains a paper bag or envelope for undergarments. Since the kit contains an envelope for the victim's reference sample (usually a buccal swab), it is important that gloves are worn and changed frequently and that each swab is dried fully before packaging. These precautionary steps are necessary to prevent contamination. In addition to collection of samples, the SANE will also take a detailed history of the assault from the victim and record it on official documents that will be kept in the case file. Depending on where the suspect ejaculated, most useful case samples are obtained from the vaginal, cervical, rectal, and oral body cavities of the victim. The victim's underwear can also be a good source of evidence since, over time, seminal fluid will drain from the vagina or anus. Other items of evidence may include clothes, bedding, blankets, comforters, furniture and car upholstery, that can be sources of seminal fluid or trace evidence, such as hairs or fibers, that may have been transferred from the suspect. Searching large items like bedding and comforters for the presence of semen stains can be hampered by detailed patterns or the color of the item. Seminal stains can appear yellowish on a white background, but may be difficult to see on a highly patterned fabric. Side lighting or special light sources can be used to locate or highlight stains. Once a semen stain has dried it may leave a crusty stain. These stains can sometimes be located by just gently touching or running your gloved fingers over the surface of the item (the crusty touch test). As before, all wet stains should be completely dried prior to packing. The investigator may outline wet stains with a wax pencil on larger items like sheets and bedding, which helps the lab analyst locate the stain once the item is back in the laboratory. Module 2: Processing Biological Evidence Page 7 Preservation of Evidence Preservation of Evidence Storage Blood and other biological evidence must never be exposed to excessive heat or humidity. Whenever possible, bloodstained evidence should be refrigerated until it can be transported to the crime lab. Evidence dried and sealed in brown paper can be safely stored at room temperature; liquid reference samples should be stored at 4°C. For further information on the collection and preservation of biological samples, please review the evidence field manual linked below, particularly section XII, The Collection, Packaging and Submission of Evidence. https://nj.gov/oag/njsp/division/investigations/pdf/ofs/Evidence_Field_Manual_201911.pdf (https://nj.gov/oag/njsp/division/investigations/pdf/ofs/Evidence_Field_Manual_201911.pdf) Chain of Custody Chain of custody is the record of evidence recovery, handling and analysis that is required by the courts for any evidence submitted in a trial. The following information must be recorded for chain of custody purposes: a description of the evidence and its container the specific recovery location of the evidence case numbers the date and time it was collected who collected it whether or not the evidence container was sealed upon transfer to another individual who received the evidence the dates and times of any evidence transfers who delivered the evidence the final disposition of the evidence By keeping a record of who has the evidence and in what location at all times, we have the best chance of maintaining the integrity of the evidence. While it is impossible to testify to what happened to the evidence while it was in someone else's custody, each person handling the evidence follows policies and procedures developed to keep the evidence secure and free from contamination. Module 2: Processing Biological Evidence Page 8 Sources and DNA Content of Biological Samples Sources and DNA Content of Biological Samples Before analysis of evidence can begin, the biological samples must be collected from a known contributor such as the victim or perpetrator/suspect or as evidence from a crime scene. The table below lists some of the types of physical evidence that can be found at crimes scenes. Type of Physical Evidence Biological Source Cigarette saliva Toothpick, dental floss saliva Stamps and Envelopes saliva Bottles, cans, cups, glasses saliva Condoms semen, vaginal and rectal cells Bedding semen, sweat, hair, urine, blood, saliva Bite marks saliva Fingernails skin, blood, sweat Tape and ligatures skin, blood, saliva Bullets/projectiles blood, tissue, skin Clothing blood, sweat, semen Hat, Mask, sweat, hair, skin/dandruff Knife, bat, other weapons blood, skin, tissue “Through and through” bullet blood, tissue Used condom Semen, vaginal or rectal cells Facial tissues, cotton swabs Mucus, blood, sweat, semen, ear wax Eyeglasses Sweat, Skin Source: Forensic analysis of biological Evidence: A lab guide for serological and DNA typing. J McClintock Whole Blood Whole blood, which is used as a reference sample for DNA analysis, may be obtained from the victim or when the suspect is in custody. Whole blood samples should be collected in a sterile tube that contains the preservative ethylenediamine tetra acetic acid (EDTA). EDTA also inhibits the activity of the enzymes known to degrade DNA. For short term storage these tubes can be refrigerated and frozen for long term storage. In some agencies the blood is spotted onto an FTA collection card which is an absorbent cellulose based paper saturated in chemicals which inhibit bacterial growth and prevent DNA degradation. FTA is an acronym for fast technology for analysis of nucleic acids. FTA papers are allowed to dry and can be stored at room temperature for years. Bloodstains and Mixed Stains When blood stained clothing is recovered then care must be taken to ensure the wet items are dry before packing to prevent contamination and cross transfer of staining which may deter the reconstruction of events. In the short term such evidentiary items can be stored at room temperature in a humidity controlled environment away from direct light. For longer term storage items must be dried and stored in a low temperature frost free (no humidity) freezer. Hairs Generally hair samples come in the form of head hair or pubic hair. 12-24 hairs collected from the scalp is usually sufficient for DNA analysis. Hair samples with intact roots provide sufficient DNA for STR (short tandem repeat) typing. A hair shaft contains enough mitochondrial DNA for mtDNA (mitochondrial DNA) typing. Hairs can be stored in the short term in a humidity controlled room at room temperature, or in a low temperature frost free freezer for longer term storage. Swabs Swabs may be obtained from wet biological material such as blood droplets, vaginal, rectal and buccal cavities. Inanimate objects may also be swabbed to recover biological samples such as cigarettes, envelopes, and bottles. Cotton swabs must be sterile and can be moistened to collect dried samples by swabbing the area of interest. Moist swabs must be dried prior to packaging or placed in a vial containing a small aliquot of sterile 1x TE buffer for short term storage. Swabs with biological material are stored in the same way as biological stains. Bone, Teeth and Tissue When circumstances result in low or insufficient in amounts of biological material such as blood and semen, for analysis, due to degradation, damage, availability or inaccessibility, the analyst may turn to bone, teeth, skin and tissue to obtain sufficient sample. Generally a section in the region of 1cm2 is enough for testing. Once samples are collected they should be frozen and transported to the laboratory on ice and kept frozen until DNA analysis begins. Note that repeated freeze thawing can greatly compromise the sample. Tissue Samples, Smears and Slides When known biological samples are unavailable but needed as part of an investigation, it is also possible to use medical samples as a source of sample. In some cases analysts have been able to obtain useful profiles from stored biopsies or stored tissue from surgical procedures including pap smears and histological sections. Usually one smear, slide or section of tissue is sufficient for successful DNA typing. These samples can be stored at room temperature indefinitely prior to DNA analysis. Although it’s labor intensive DNA can also be isolated from paraffin embedded tissues and the yield is often low and impure. Recently xylene has been used for paraffin removal which eliminated some extra handling steps that have been attributed to sample loss in past methods resulting in a slightly higher sample yield compared to earlier processes. Semen and Sperm Sperm collected from a vaginal swab will also contain epithelial (skin) cells from the female and possibly the male. In these situations the sperm can be differentially separated from the skin cells using extraction methods. When samples are thought to contain only spermatozoa, such as ejaculate recovered from a used condom, the sample can be collected and processed in the same way as described above for bloodstains or mixed stains. Sperm specimens should be stored frozen prior to analysis and multiple free thaw cycles should be avoided. Urine Urine is similar to other biological samples such as sweat and sebaceous oils, in that once it is concentrated it will contain a sufficient amount of epithelial cells to generate a DNA profile. A minimum volume of 10ml will generally be sufficient, but when available as much as 30 ml would be optimum. Urine samples should be stored frozen and refrigerated for a short period of time prior to analysis, again avoiding multiple freeze-thaw cycles. Concentrated samples can also be collected from a stain or a swab from a known source. Fingernail Clippings Fingernail clippings can be a great source of biological material for DNA analysis. Clippings collected from the victim or crime scene can be used to identify a suspect as well as predict the gender of the attacker. During a sexual assault the victim may scratch the attacker while defending themselves and collect skin and blood under their fingernails. Nail clippings and the underside of nails can be swabbed using a sterile, moistened cotton swab. The swab is dried and placed in a container for storage or placed in a vial containing an aliquot of 1x TE buffer for short term storage prior to DNA analysis. Swabs can be stored long term in the way as stains. DNA Content of Biological Samples Sample/ Source DNA Content ( approx) Liquid blood 20-40 ug/ml Blood stain 250-500 ng ( from 1 cm2 stain) Semen 150,000-300, 00 ng /ml Post Coital Vaginal Swab 0-3000 ng/ml Saliva 1000-10,000 ng/ml Oral Swab 1000-1,500 ng/ml Hair (root) 1-750 ng/ plucked root Hair (Shed) 1-12 ng/hair Urine 1-20 ng/ml Bone 3-10 ng/ml Tissue 3-15 ug/mg Fibroblasts 6.5ug/1x106 cells Source: Forensic analysis of biological Evidence: A lab guide for serological and DNA typing. J McClintock. Biological Evidence Overview Power Point Presentation - Click Here! (https://ufl.instructure.com/courses/469766/files/72988786/download?wrap=1) Biological Evidence Overview - PDF File (smaller size/faster download) - Click Here! (https://ufl.instructure.com/courses/469766/files/72988785/download?wrap=1) Module 3: Laboratory Analysis of Biological Evidence (Blood) Page 1 Module Overview Introduction When items of evidence are submitted to Forensic Serology, the first thing the analyst must do is inventory all of the items of evidence and decide which would be most probative for the investigation. Sometimes this task is rather easy for simple cases with a minimal amount of evidence. Other times, cases may be comprised of hundreds of items of evidence from multiple crime scenes. In busy laboratories, it is impossible to process each item of evidence, therefore the Forensic Serologist must be skilled in case management and choosing which stains on which items would be the most likely to yield meaningful results. Once the analyst has decided which pieces of evidence to process, they must begin the process of screening the evidence. Since it is important to avoid cross contamination, evidence from different crime scenes and evidence from different people must be processed in different secured locations. Laboratories may have multiple separate rooms or examination areas for serologists to work in, allowing them to switch to different rooms for items of evidence from different sources. Additionally, the serologist must take care to change their disposable gloves between each item of evidence and change their entire regiment of PPE (including gloves, hair cover, mask, lab coat) between each source. Prior to examining evidence, the analyst will be sure to sanitize work surfaces, tools and writing utensils. This practice prevents the chance of genetic material contaminating a case that it is not associated with. Concluding visual examination of the evidence and taking detailed notes describing it and the condition it is in, the serologist will begin the process of presumptive testing. These tests are rapid chemical color tests that require very little sample and provide an indication of the nature of the stain. These tests are called presumptive tests and cannot be used to positively identify the nature of a stain since they also react to produce a color change with several other substances. Once a stain is tentatively identified, further confirmatory microscopic or chemical tests may be conducted to unequivocally identify the stain. In some instances, biological evidence may not be present or may not be visible to the naked eye. Indirect light sources and stereomicroscopy can be used to take a closer look at evidence, sometimes revealing tiny biological stains. Additionally, if no stains can be located, mapping techniques can be used to generalize areas of presumptive positivity. In the event that no staining is located, the serologist should not feel like they missed something. Evidence can and sometimes will be negative! In this module, you will learn about the tools and techniques that are used in the lab by forensic analysts to find biological evidence. Objectives At the completion of this module, students should: Understand the process used in the laboratory for the searching and recording of biological evidence Understand commonly used techniques to find blood, in forensic casework Understand the issues involved in multi-section cases Understand the significance of false positive and false negative tests Understand the specificity and sensitivity of the tests Understand the difference between preliminary and confirmatory tests Understand the chemistry of preliminary tests and screening tests for blood Module 3: Laboratory Analysis of Biological Evidence (Blood) Page 2 Searching and Note Taking in the Laboratory Searching and Note Taking in the Laboratory The following examination procedure can be applied to most forms of evidence encountered by the forensic biologist, including items of clothing, bedding, some kinds of weapons, fabrics, textiles and items recovered from scenes, and vehicles. Searching and Note taking The analyst selects an isolated area to examine the evidence. Wearing a clean lab coat, the bench area is covered with a clean sheet of paper prior to opening any evidence bags (this will catch any debris that falls off the item being examined) - this paper is changed prior to the examination of each item. Packages of evidence are checked against the evidence list in the case file; where necessary, evidentiary items are divided into those obtained from the suspect, those from the victim, and those from the scene. Evidence from different sources such as suspect, victim, and scene, should be examined in separate areas/rooms to avoid cross-contamination of trace materials (hairs, fibers, etc). Once an item of evidence is selected for examination, the evidence bag is opened carefully in a manner that preserves the original seal and signature. The contents are carefully removed onto the paper, and the bag is checked to recover any trace evidence that may be lost in the packaging. While making notes at each step, the item is sketched and carefully searched; obvious debris and potential evidence is recovered, recorded and packaged; fibers and hairs are mounted on glass slides and other trace evidence is packaged and submitted to the appropriate section for analysis (pieces of glass or paint may be submitted to the appropriate section (chemistry, criminalistics) for analysis. The appearance and location of any stains or damage on an item are noted in the records as well as the location of any trace evidence that is recovered from the item under examination. Pre-drawn templates can be used for recording information on commonly examined items (such as articles of clothing). Notes can reflect the characteristics of the item such as color, manufacturer, size, any tears/damage, stains, etc. which makes it easy to identify in court. Presumptive tests are carried out on suspect stains. If stains present on the clothing are negative, remainder of item needs to be swabbed and tested as well since not all stains are visible to the naked eye. Areas of stain that test positive with presumptive tests are selected for confirmatory tests and DNA analysis; Genetic differences between a suspect stain and a reference sample taken from the possible source allows that source to be positively eliminated (this source could be a victim or an assailant). Once examination is complete, the item of evidence is wrapped is wrapped in the exam paper it is opened on so as to make sure that no trace evidence is inadvertently discarded. Module 3: Laboratory Analysis of Biological Evidence (Blood) Page 3 Blood Blood Composition of Blood Blood is a complex mixture of cells, enzymes, proteins and inorganic substances. The fluid portion of blood, the cell free fraction or plasma, is mainly water and accounts for about 55% of blood content. The solid materials, that are mainly cellular, are suspended in the plasma. There are three main types of blood cells that make up about 45% of the total volume of blood: Red blood cells (RBC), or erythrocytes, are unique in that the mature circulating cells have no DNA. These cells function to provide oxygen to tissues by transporting oxygen molecules as part of a hemoglobin complex. The total volume of red blood cells is called the hematocrit. White blood cells (WBC) or leukocytes are involved in the body's responses to infection. One group of lymphocytes are responsible for antibody production. WBC contain DNA. The third kind of blood cell is the platelet. These blood cells are involved in the blood clotting process: blood clots when of one of the dissolved proteins, fibrinogen is converted to a polymer called fibrin that traps platelets to form blood clots. The liquid that remains when blood is clotted is called serum. There are several different serum proteins, but about half consist of albumin. Albumin plays a major role in preserving blood volume by regulating osmotic pressure. The remaining serum protein, which include molecules such as antibodies are classified as globulins. The main salts circulating in blood are sodium, potassium and chloride ions, and other organic substances such as glucose, hormones and vitamins. Blood circulates throughout the body through the blood vessels that make up the circulatory system. The high pressure arterial system, that helps propel the blood from the heart to the major organs of the body, is composed of vessels with strong elastic walls. The venous system that returns the blood to the heart is much less elastic and under much less pressure. It is this pressure and the structure of the blood vessel that determines whether blood oozes or spurts from an injury site. Damage to a high-pressure artery or arteriole can result in the characteristic arterial spurt. Module 3: Laboratory Analysis of Biological Evidence (Blood) Page 4 Locating Stains Locating Stains Blood stains typically have a reddish brown appearance. However, blood stains can take on many appearances on different items of evidence. Blood may look different on a red fabric vs. white fabric vs. black fabric. The stains may be old and degraded and may have a yellowish-greenish appearance. Some stains may have been exposed to the environment and be mixed with dirt. The stains may have been washed and appear lighter than a fresh blood stain. All of these scenarios are commonly seen in routine forensic casework. Although blood does not fluoresce under an alternative light source like other body fluids do, there are tools available to aid the examiner in looking for stains. Illuminated magnifiers are large magnifiers with a light source attached. This can aid in looking at items such as guns or knives, where the blood may be in cracks and grooves and not readily seen with the naked eye. It is also helpful on black or dark clothing since the dark fabric may mask the stains. Infrared lighting can cause many fabrics to reflect a large amount of the light. However, bloodstains generally absorb most wavelengths of visible light as well as near infrared wavelengths of 700 to 900 nm. This results in the fabric appearing gray or white and the bloodstains dark in color which can be seen clearly in black and white infrared photographs. Other tools such as a simple flashlight or a stereoscope can also be used. The first step in processing an item for the presence or absence of blood is to perform a visual exam. Any areas of interest should be marked (if possible). If it is necessary, illuminated magnifiers or other tools may be used to aid in locating the stains of interest. Next, each individual stain should be tested using the laboratory’s chosen presumptive test for blood (such as Kastle-Meyer, Leucomalachite Green etc). Sample can be obtained by rubbing the suspect area with a dry swab. The same method can be used to locate blood hidden in cracks such as between the handle and blade of a knife. Although very little stain may transfer to the swab the tests are sensitive enough to locate the presence of possible blood. All results should be recorded on the item and in the notes. It may be beneficial to draw a diagram of the item or take a digital picture where the positive areas can be marked and used for later DNA characterization. This documentation is also very beneficial in court if you have to testify to your findings. Sometimes where the stain is on an item of evidence can play an important role in the case. If all areas tested are negative OR the item you examine has no visible stains of interest, a general mapping / swabbing technique can be used. This is done by sectioning off the item and taking a general swabbing from each section and testing each one. Each area of the item should be swabbed individually ( for example, front of sweater, back of sweater, front of right sleeve, front of left sleeve etc) in order to be able to narrow down an area when positive testing occurs. If there is a positive reaction to one of these swabbings, then the examiner has narrowed down where that stain is on the item. From there, smaller sections can be made to isolate the exact stain. If you are concerned about exhausting the stain by performing more testing, the general area can be swabbed and the swab sent on for further characterization. Once a possible blood stain has been identified, a portion of the stain (or all, depending on the size) can be removed from the original item for further characterization. In many laboratories, the removal of a stain is witnessed by a second scientist. The reason for this is to double check the notes and sketch depicting where the stain was removed from as well as to check the labeling of the packaging for the removed stain. Typically, the removed stain will be sub-labeled from it's parent item. For example, if a stain is removed from item #3, a tshirt, the sub-labeled stain will be called #3-1 and the witness will verify that the case number and sub-item number on the new envelope containing the stain matched the analysts notes. Common Collection Techniques The type of collection technique you should use to remove a small portion of a stain for further testing will depend on the item that the stain is on. Some common collection techniques are listed below with examples of the types of evidence they can be used on. Cuttings - Fabric (ex. clothing) or other soft, porous surfaces that can be cut (ex. paper). Swabbings - Hard surfaces (ex. guns, knives) or fabric (especially fabric where the stain is easily swabbed like nylon or leather). Scrapings -Fingernails (scrape the underside surface for possible foreign debris or blood from an attacker or victim), and hard surfaces (if the blood stain is heavy it can scrape off in flakes). Chemical Tests Module 3: Laboratory Analysis of Biological Evidence (Blood) Page 5 Chemical Tests Presumptive Tests Presumptive tests are sensitive but not specific tests for body fluids that depend on the detection of constituent chemicals. Presumptive blood tests are rapid, chemical tests that may be used to locate and differentiate between blood and other similarly colored stains. Presumptive tests are highly sensitive, easy to perform and allow minute traces of blood to be located when they might not be easily noticed. They are sensitive enough to apply to areas of scenes that may have been cleaned in an attempt to hide the evidence. Most presumptive tests for blood rely upon reactions associated with the peroxidase activity of hemoglobin. The most common tests depend on the oxidation of colorless reduced chemical indicators. Most presumptive tests are color tests where the reaction with blood results in a color change in the reagent. Unlike the other color tests, the stain visualizing enhancer Luminol causes stains to chemiluminesce Common Color Tests for Blood Test Indicator Sensitivity Benzidine Blue color ++++ Tetra methyl benzidine Blue color +++ o-Tolidine Blue color +++ Phenolphthalein or Kastle Meyer test Pink color ++ Leucomalachite green Green color ++ Luminol (5-amino-2,3-dihydro-1,4 phthalazinedione) Fluorescence +++++ These presumptive tests are not specific for blood, since other substances possess peroxidase activity. These include chemical oxidants such as rust and vegetable peroxidases such as horseradish. The most commonly used presumptive blood tests for blood include the Kastle-Meyer test (Phenolpthalein), and LMG (Leucomalachite Green). Because of the possibility of false positive reactions with other oxidizing agents, the Kastle-Myer test can be performed as a 3-step test. A swab is rubbed on a small section of the stain and that swab is then tested. This is called an indirect test since chemical reagents are not applied directly to the evidence. Step 1 is the addition of a few drops of ethanol, which increases the sensitivity of the test. Step 2 is the addition of a few drops of phenolphthaliein after which the analyst will pause for a few seconds to observe any color change. A change of color from clear to pink at this stage means that an oxidizing agent may be present (such a plant peroxidase). Step 3 is the addition of a few drops of hydrogen peroxide which, in the presence of hemoglobin, oxidizes colorless reduced phenolphthalein to pink phenolphthalein. Of course, it should be noted that the reagents should be tested using known blood every day to ensure they are working properly. More recently the Hemastix test is also being used at crime scenes due to its ease of use. One thing responding officers should keep in mind when using chemical tests is the pros and cons of field testing. While hemastix may be useful in deducing which samples to collect, samples are typically tested again in the laboratory for the scientist to document results. In addition to multiple tests possibly reducing the likelihood of obtaining a good DNA profile, the officer may be asked to testify to the science behind presumptive testing. For these reasons, field testing is sometimes discouraged in favor of laboratory tests. KASTLE-MEYER Commercial Kits Many presumptive tests are commercially available in kit form for rapid, easy and low risk fieldtesting. Most field test kits for blood test contain the testing agent Luminol and one or two other presumptive test reagents in disposable chemical applicators. Kits also include test papers, sterile water, instructions and a color chart. Module 3: Laboratory Analysis of Biological Evidence (Blood) Page 6 Confirmatory Tests Confirmatory Tests Most modern forensic laboratories do not employ confirmatory tests for blood. Since DNA typing became routine, many laboratories simply couple a presumptive test with DNA typing instead of using a traditional confirmatory test for blood which may consume more sample and time. Since DNA kits have a probe for human DNA, they will not amplify animal blood or other substances. However, it is important to remember that a presumptive test combined with DNA typing is not a confirmation of human blood. Should this confirmation be needed, there are several traditional tests that can be used. Microchemical Tests To confirm the identity of a positive presumptive test for blood, there are two microchemical tests that can be used to confirm the presence of blood through the production of characteristic crystals. The most common test is the Takayama or hemochromogen test. In this test, ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin. Once the reaction occurs, the crystal product can be observed using a light microscope. The other confirmatory test is the Teichman reaction that works through the same principles as the Takayama test. Although both tests give comparable results, the Takayama test tends to be preferred, probably because it's easier to use and the same characteristic crystals tend to form with a range of stain types. These tests tend to be less sensitive than color tests and can be disrupted by dirt and other particulate matter present in the sample. Microscopy Sometimes laboratories will use microscopy to verify the presence of blood cells in a suspected blood sample. While this is a straightforward process when dealing with whole blood, it's much more difficult when dealing with dried bloodstains. With dried stains, the cells must be reconstituted, re-dried, fixed and stained for microscopy for the identification of characteristic morphological features. Successful identification using this technique really depends on the level of expertise of the crime lab analyst and their ability to find intact cells within the stain. The integrity of dried red blood cells can be affected by the age of the stain, the condition of a stain and the nature of the surface the stain is on. Spectroscopy Other tests for blood rely on the use of spectroscopy to distinguish between hemoglobin and hemoglobin derivatives such as methemoglobin, carboxyhemoglobin, sulfhemogobin and oxyhemoglobin. This is considered to be a reliable technique that has been shown to give results with older stains that have tested negative using color or microcrystal tests. All hemoglobin derivatives exhibit a strong absorbance at 400-425 nm (called the Soret Band). Hematrace Another commonly used “confirmatory” test for the presence of blood is the ABAcard Hematrace test. This is a test that detects the presence of human hemoglobin in a sample. It is based on an antibody-antigen reaction between the sample (hemoglobin = antigen) and the antibodies in the test card. This test works much like a pregnancy test and looks very similar. Not all labs use this test because the Hematrace card has been shown to react positively with ferret blood. For this reason some laboratories will not use it as a confirmatory test for human blood. Other laboratories still use this test to confirm human blood but may put a disclaimer in the lab report indicating it’s cross reactivity with ferret blood. Module 4: Laboratory Analysis of Biological Evidence (Semen and Other Biological Evidence) Page 1 Module Overview Introduction Continuing from the previous module, we will focus on the processing and forensic testing of semen and other biological evidence. We will outline the most widely accepted techniques and processes for examination of evidence for the presence of semen and we will introduce you to the searching techniques and presumptive and confirmatory testing of seminal fluid and the identification of spermatozoa. This module will also outline the available tests for remaining types of biological evidence Objectives At the completion of this module, students should: Know the available tools that are used in laboratories to aid in locating stains of interest Understand commonly used techniques to locate and test semen, saliva and other biological fluids in forensic casework Understand the issues involved in multi-section cases Module 4: Laboratory Analysis of Biological Evidence (Semen and Other Biological Evidence) Page 2 Semen- Searching Techniques Semen - Searching Techniques Semen is the biological fluid produced by the male sex organs. Semen has a complex composition made up of a cellular component, the spermatozoa, and a fluid component, the seminal plasma. An average ejaculate is 2 to 6 ml and contains 100 to 150 million sperm. Sperm are the male reproductive cells. Each consists of a head, tail and middle. In humans, the head is a tiny disc, about 4.5 µm long and 2.5 µm wide and contains the DNA. The tail is about 40 µm long, and tends to be rapidly lost after ejaculation. Ape sperm are similar in size and shape to human sperm. Dogs have similar shaped sperm but are different in size to human sperm. Other animals have different shaped sperm that are characteristic to their species. It is important for a Forensic Serologist to view different animal sperm during training to be sure they can readily recognize non-human sperm during casework. Seminal plasma contains, cellular materials, proteins, salts, organic substances such as flavins, which are the source of its UV fluorescence, and choline. Some components originate from glands and organs such as the seminal vesicles and the prostate gland, which is the source of acid phosphatase and p30 protein. In vasectomized males, the surgical procedure involves severing or ligating the ducts carrying sperm to the penis. Therefore vasectomized men will have no sperm but will have the plasma components present in their ejaculate. After ejaculation, semen is lost from the vagina over time, through the processes of drainage and biochemical change. The tails of the spermatozoa are lost first - the damage beginning immediately after ejaculation. After about 6 hours, about 25% of the spermatozoa will have no tails and by 12 hours, there will be few sperm with intact tails. Twenty four hours after ejaculation there are usually only a few heads left, however these proportions and times are quite variable. The survival time of sperm in stains outside the body depends largely on environmental conditions, but a small stain that has dried quickly may have intact sperm preserved for months or even years. Visual and Tactile Examination Semen stains on items of evidence can often be visible to the naked eye. Semen dries to form a white stain on dark surfaces, but may be more yellow in color if dried onto a white surface, or if the stain is old. On either surface, the stain may form a thin crust that imparts a rough or granular texture to the stain. On darker surfaces stains are often easy to locate with the naked eye, however on more patterned surfaces it may be necessary to use an alternative light source to highlight stains or to use a tactile test (crusty touch test) by gently running your gloved fingers across the surface to locate the crusty stains. Stains not visible to the examiner may be located using additional tools. However, a visual exam should always be performed first, and any suspect stains indicated on the item. This can be done by marking directly on the evidence with a wax pencil. ALS After a preliminary visual and tactile examination, an Alternate Light Source (ALS) may be used to locate less obvious staining. ALS is particularly useful on large items such as bedding or in vehicles. Semen has fluorescing properties due to bacteria (pseudomonas fluorescens) and flavins that are present in semen. The fluorophores present are excited by an energy source (light from the ALS) and emit a light energy rendering the stain visible. Semen is typically visualized best using 400-500 nm wavelengths and using orange safety goggles. An ALS exam must be done in a dark room so that the fluorescence can be seen. Keep in mind that other body fluids such as sweat, urine and saliva can also fluoresce so an ALS exam is not an actual test for semen, it is a searching tool. Blotting Blotting can be useful on items such as clothing and bedding. Larger items may be searched for stains using a mapping process. Large sheets of damp blotting paper are systematically applied to the item. Papers are applied following a grid like pattern and labeled to indicate their location on the item of interest. Papers are pressed against the item, then removed to a fume hood and sprayed with the color test reagent. This form of searching is used to determine whether acid phosphatase is present and to determine the distribution of staining on an item. Applying the damp blotting paper against the item results in wetting the original stain and a high risk of transferring the original stain to new areas, especially where layers of fabric overlay each other under the blotted area. To prevent further transfer the analyst should place a sheet of polythene between the upper layer of fabric being tested and the layers underneath. This process can also be done with items of clothing, underwear and large items of bedding. In addition, the analyst must ensure that the blotted item is fully dried before repackaging. Once the location of all stains have been identified, the analyst will then begin confirmatory testing on these areas. Each stain that is mapped is normally marked with a wax pencil. The forensic serologist will then take a small clipping of each AP (acid phosphatase) positive area and place it into a small tube with a few milliliters of distilled water in preparation for microscopy. All results should be recorded on the item and in the notes. Presumptive Testing Acid Phosphatase Spot Test Human semen contains very high concentrations of acid phosphatase, which is an enzyme secreted by the prostate gland. In the body, acid phosphatase is responsible for cleaving a phosphate from phosphorylcholine yielding choline which is important in cellular membrane composition and repair. Presumptive tests for semen are based on the hydrolysis of phosphate esters and the detection of a liberated organic colored complex. The most common test for acid phosphatase is called the AP test. Acid phosphatase, if present in the sample reacts with a chemical called sodium alpha-naphthyl phosphate to form naphthol. Naphthol when combined with Fast Blue B produces a purple azo colored product. This test can be performed in a one step process or a two- step process. In the one step process, the sodium alpha-naphthyl phosphate and the Fast Blue B are combined into one reagent which is applied to a sample of the stain in question. A purple color change within 60 seconds (depending on the lab protocol) indicates the possible presence of acid phosphatase in the sample. The two step process differs in that the sodium alpha-naphthyl phosphate (reagent A) and the Fast Blue B (reagent B) are used as separate reagents where one drop is applied of reagent A, then a drop of reagent B to a sample of the stain in question. The same color change reaction is expected with the two step process. BCIP Test Another presumptive test for acid phosphatase is the BCIP test (5-bromo-4-chloro-3indolyphosphate). This test is performed by taking a swabbing of the stain in question and adding it to a tube filled with BCIP solution. If acid phosphatase is present it will hydrolyze the BCIP creating a blue color change within ~ 15 minutes. Both the AP spot test and the BCIP test are presumptive in nature because of the many false positives associated with these tests. Other body fluids can contain other forms of acid phosphatases that may give a false positive reaction such as vaginal fluid (vaginal acid phosphatase is elevated in prepubescent females) and blood (erythrocytic acid phosphatase and seminal acid phosphatase can be elevated in males with prostate cancer). Also, male urine may have acid phosphatase present. Other substances such as plant matter, feminine hygiene products, and spermicides can also give a false positive reaction with the AP tests. With most forms of biological evidence, a negative reaction with a presumptive test would generally end further testing of that stain. Items in a sexual assault kit (swabs from vagina, cervix, anal, oral and also panties) should be confirmatory tested regardless of the presumptive test results. This is due to the fact that sperm can last longer in the body cavities than acid phosphatase. Resources Reference information for seminal fluid can be accessed at the following site: http://www.news-medical.net/health/Semen-What-is-Semen.aspx medical.net/health/Semen-What-is-Semen.aspx) (http://www.news- Module 4: Laboratory Analysis of Biological Evidence (Semen and Other Biological Evidence) Page 3 Semen Continued Confirmatory Tests Microscopy All AP or BCIP positive samples should undergo microscopic examination to confirm the presence of spermatozoa. The most reliable technique for semen identification is histological staining and light microscopy, since the morphology and dimensions of the human spermatozoon are unique. However, the small sperm can be difficult to locate even using microscopy, since many will have lost their characteristic tails and the heads may be mixed with bacterial and yeast cells present in the sample. In order to prepare a sample for microscopic analysis, the serologist will cut a tiny piece of a swab from the sexual assault kit of fabric from clothing or bedding and place that tiny cutting into a micro centrifuge tube with a bit of distilled water. The tube then agitated and centrifuged, creating a pellet of cells in the bottom of the fluid. The pelleted cells are then applied to a glass microscope slide and heat fixed (dried) before staining. The most commonly used stain in the US is Christmas tree stain - named so because of the bright colors it produces. The staining solution includes Nuclear Fast Red to stain the DNA-containing heads bright crimson, and a counter-stain of picric acid indigocarmine (PIC) that stains the tails green. Traditional histological staining with Hematoxylin and Eosin (H&E) is also used, but tends to be more common in European crime lab systems. H&E stain will stain the nuclear material (sperm head) purple and the non nuclear material (tail) pink. A newer type of stain is a fluorescent stain called SpermPaints that is a fluorescent dye mixed with antibodies which bind to the sperm head and tail antigens. This stain is clear, bright and selective only for sperm cells and will not stain any other debris or cells in the sample. This type of stain is able to be used in conjunction with laser micro-dissection microscopes. These microscopes automatically find sperm cells on the prepared slide and then have the ability to dissect out only the sperm cells using a laser, isolating these cells in a centrifuge tube for later DNA processing. A test sample is considered to be positive for semen when any number of intact heads and tails are observed on the slide. The presence of one or more heads and no visible tails may also be considered to be a positive result in some laboratories, depending on their policies and criteria for positive sample determination. The issue of intact (with tail attached) vs non intact (without tail attached) sperm has been debated over the years, however, a spermatozoon with or without a tail is still a spermatozoon and any limitations to this means of positive identification lies with the ability of the analyst to identify non intact sperm. During microscopic examination, you are always looking at the differential staining for the presence of the acrosomal cap on the head and a tail (if attached), the morphology in general of the sperm and its size. Human sperm cells are larger than yeast and bacteria but smaller than free nuclei (from epithelial cells) and dog sperm. Dog sperm are similarly shaped and stain the same as human sperm but are approximately 3 times the size of human sperm. If, with any piece of evidence, you obtain a positive presumptive test, further positive identification problems may be encountered if the seminal fluid is from a man who has a low sperm count (oligospermic) or who has no spermatozoa present in his seminal fluid (aspermic). When the presumptive Acid Phosphate test indicates the presence of semen, but the microscopic analysis shows no detectable spermatozoa, further tests are carried out to determine the presence of the protein, p30 or prostate specific antigen (PSA) which is only found in high concentration in human semen and is secreted by the prostate gland. It is also called P30 due to its protein molecular weight of 30,000 kD. The function of this protein is that it liquefies semen and aids in dissolving the cervical mucus cap to aid in sperm entry. Prostate Specific Antigen (PSA)/P30 PSA, or P30, can be detected by precipitin reaction with a specific antiserum using the Ouchterlony process (more on that later). There is also a quantitative immunological test utilizing an enzymelinked reaction (ELISA). The type of tests most often used today to detect the presence of PSA is based on an antibody-antigen reaction between the sample (PSA) and the antibodies in the test card. It works very similarly to the test previously described for the detection of human hemoglobin (Hematrace®) There are two main test cards that are used for this type of testing: the ABAcard ® P30 test and the Seratec ® PSA Semiquant. Both test cards work similarly, however the Seratec test card has a semi quantitative internal control (set at 4ng/ml) that aids in detecting semen concentration. Testing for P30 may be considered to be a confirmatory test for the presence of semen and is important to use in cases of a vasectomized or aspermic individual to confirm semen when sperm cells would not be present in the sample to confirm via microscopic analysis. P30 can be present in the blood of males with prostatic cancer and also male urine but is not expected to be found in as high concentrations as can be found in semen and would not be detectable with this type of test. Resources Below: microscopic images of intact spermatozoa. TSI Interval The question of how long was the sperm in the vagina is consistently raised during case investigations or in court. The time since intercourse (TSI) interval is a parameter used to determine whether recovered semen is due to the reported assault rather than from a consensual sexual act that occurred sometime before the alleged assault. Studies have been conducted to investigate the time it takes seminal constituents to leave the vagina and other body orifices, but the results are unclear and difficult to interpret. In most cases, forensic labs don't obtain orifice swabs for analysis in enough time to see motile sperm, but data obtained from hospitals have shown that sperm survive in the vagina for about three hours after ejaculation. However the survival time actually ranges from 1 to 8 hours depending on conditions. Motile sperm seem to survive longer in the cervix and may survive for several hours or several days. Intact spermatozoa have been shown to persist in the vagina for up to 26 hours, and heads for up to 3 days. On rare occasions heads have been seen to persist maybe as long as 7 days. If abundant spermatozoa can be seen in every microscope field then the swab was probably obtained a day or less after the incident. If the spermatozoa are very few or difficult to find on the slide then intercourse could have occurred up to five days earlier. It must also be remembered though that the presence of spermatozoa can also depend on whether full or partial penetration occurred; whether there was complete ejaculation by the assailant; variations in sperm count, and the victim's activity before or after the incident. Spermatozoa survival in the rectum of living victims has been estimated to be 6 to 65 hours and survival in the oral cavity tends to be much shorter, in the region of 6 hours, since saliva and drinking fluids wash away the sperm. The level of acid phosphatase in the post coital vagina has been shown to persist for 6 hours to 3 days, and using ELISA techniques or P30 antibody/antigen test cards, most p30 has been shown to be eliminated within 24-27 hours of intercourse. Since there are so many variables in determining TSI, it is more prudent to obtain reference samples from the suspect (if known) and elimination reference(s) from any consensual partners the victim may have had. Module 4: Laboratory Analysis of Biological Evidence (Semen and Other Biological Evidence) Page 4 Saliva Saliva Saliva is an alkaline fluid that moistens the mouth, softens food, and helps digestion. There are three glands that produce saliva that are located around the mouth under the lower jaw, beneath the tongue, and in front of each ear. These glands are the sublingual, submandibular and the parotid glands. Buccal glands, in the cheeks near the front of the mouth, also secrete saliva. The saliva of the parotid gland contains enzymes called amylases that facilitate the digestion of carbohydrates. Saliva stains are fairly difficult to locate since they leave a very faint trace when dried and typically cannot be seen with the naked eye. An ALS exam can be performed, but saliva may fluoresce to a much lesser extent than semen. The test for detecting saliva is the location of the salivary enzyme alpha amylase. Saliva screening depends on the ability of the amylase enzyme to split water-insoluble amylose that has been covalently bound to a dye, to soluble saccharide products that result in a color change. Again, other bodily fluids such as vaginal fluids, breast milk, sweat, blood serum, semen and fecal material may contain some amylase in low concentrations that can result in a weaker test reaction than salivary amylase, producing a false positive result. The best screening technique (amylase mapping) is similar to that used in searching large items for seminal fluid, using large sheets of blotting paper that have been stained with the test reagent. The dried paper is then moistened with water and pressed onto the area to be tested. The item is then searched in a systematic manner using fresh paper on each area to be tested. The most common reagent used to identify amylase is Phadebas, which are tablets that consist of a blue dye cross -linked to starch (β1-3 glucose bond). In the presence of amylase, the starch is digested, releasing the dye into solution resulting in a blue coloration of the area. A similar test involves the use of Procion Red MX2B amylopectin, also called Lyosine red. In this case the reagent is pink and if the paper comes into contact with saliva, within a few minutes the stain will cause the pink paper to become white in the contact area. Positive tests will infer, but not confirm the presence of saliva The Phadebas test can also be used quantitatively which may be more sensitive and specific than the mapping process. Using this method, the amylase substrate is cross -linked to starch and made into a tablet. Amylase present in a sample hydrolyses the polymer to soluble blue starch that can be measured colorimetrically at 620 nm. An absorbance value that exceeds 0.3 absorbance units is considered a positive reaction. Another test for detecting amylase in a sample is the radial diffusion test. This test can give false positive results, especially with mixed fluid stains. The test uses an agarose gel containing 1% starch. An extract of an unknown sample is added to a small hole punched into the gel and allowed to diffuse radially. Amylase present in the sample will hydrolyze the starch in the gel as it diffuses and will produce a clear area on the gel after staining with iodine solution. The size of the clear area is directly proportional to the amount of amylase in the sample. An alternative to mapping is to take general swabbing from an item where saliva may be indicated from a case scenario OR taking representative cuttings from these areas. Collecting saliva from bite marks, especially those that are readily visible, is a simple process that involves wiping the area with a moistened cotton swab. The swab can then be air dried and then tested. Bites that do not occur directly on the skin but are inflicted through clothing tend to be a bit trickier. An alternative to mapping is to take general swabbing from an item where saliva may be indicated from a case scenario OR taking representative cuttings from these areas. For example, if the person was bitten on the right breast, samples can be taken from that general area. Saliva analysis may also be useful in cases such as burglary where a full face ski-style mask was recovered. The analyst may prefer amylase mapping to pinpoint where saliva was deposited inside of the mask. This is important because the location of a saliva stain may contain a high enough concentration of epithelial cells to develop a DNA profile. Many laboratories do not have procedures for amylase testing. This may due to a number of factors. Saliva is difficult to isolate so not being able to visualize it does not mean it isn't there. People often have varying levels of amylase activity and while one person’s saliva may react with an amylase test, another’s saliva may give inconclusive or negative results. For these laboratories, a sample is taken from an item of evidence if the case scenario indicates saliva may be present and the sample is extracted for DNA testing. Saliva in Sexual Assaults Amylase testing may be considered for certain types of sexual assaults. Saliva is often used as a lubricant in juvenile sexual assaults and may be detected (and further characterized) on underwear. Other situations include oral assault or if the victim was unconscious and does not remember what happened during the sexual assault. Such tests are generally of no value if the victim has showered prior to sample collection. Prior to screening an item of evidence, it is important to look at the case scenario and talk to the detective about what is needed in the case. Often, trace evidence and hair are important. Many laboratories have separate trace analysts who sweep or remove evidence such as hair, paint ships, soil, gunshot residue, or fibers PRIOR to biological screening to as to avoid the loss of such evidence in the manipulation of the item. If the laboratory does not have a separate analysis for trace evidence, then you must remove this evidence prior to screening the item and place it in a debris fold for possible later analysis. Urine, feces and vaginal secretions may also be encountered in sexual assaults, sexually motivated homicides, and incidents involving sodomy or bestiality. Module 4: Laboratory Analysis of Biological Evidence (Semen and Other Biological Evidence) Page 5 Urine and Feces The following are less common tests that some laboratories may utilize. These tests are unlikely to contain enough DNA to develop a profile and therefore may not be as useful in criminal cases. Urine Urine is the net filtrate from the kidneys, characterized by high concentrations of urea and creatinine. Urine has a pale yellow color that comes from the pigment urochrome, a product of urobilinogen or urobilin. Visually locating a urine stain on many surfaces and fabrics can be difficult, but urine does have a very distinctive odor. The odor is ammonia-like and is a result of the bacterial breakdown of urea. Alternative light sources can be useful in finding urine stains since urine fluoresces faint bluewhite. Chemical tests One type of urine screening test depends on colorimetric detection of ammonia produced from urea by urease. There are no confirmatory tests for urine; the presence of urine can only be inferred from a positive result obtained from the presumptive test. The urea litmus paper test indirectly indicates the presence of urea by reacting the sample to urease which would generate ammonia from the urea. The ammonia, which is a basic chemical, can then be indicated using a litmus paper. Another test based on the same reaction is called the Urea-nitrogen test where the ammonia is indicated by the production of a deep blue color reaction. Urea + H2O ↔ CO2 + 2NH3 Another type of test, which is more commonly used to indicate urine is the Jaffe test, which detects creatinine, a breakdown product of creatine (a component of muscle), that is eliminated through the kidneys. Using the Jaffe test, creatinine forms a red compound with the addition of picric acid. Microscopy Microscopic examination of urine relies on the fact that urine contains several solid materials with characteristic crystalline structures, as well as the presence of epithelial cells characteristic to the urinary tract. Feces Feces are the unabsorbed residues from the gut. They include food residues, gut wall debris, digestive secretions, and bile pigment breakdown products such as urobilinogen. Chemical tests Feces have a characteristic odor that is mainly due to the chemical compound skatole (which at low levels smells good and is a component of perfumes and colognes). Urobilinogen is a bile pigment excreted in feces and reactions with this compound form the basis of presumptive tests for feces. Urobilinogen can be oxidized to a pink-red product with mercuric chloride; using a 10% solution of mercuric chloride in amyl alcohol as reagent, the suspended sample is centrifuged and the supernatant is treated with zinc chloride. This test became known as the Edelman test, although its reliability has been disputed over the years. Microscopy Microscopy is one of the oldest methods for identifying fecal matter. A sample of feces will contain the undigested residues of food digested within the last 12-24 hours. An analyst with sufficient experience can identify characteristic fibrous materials that are found in animal meats, fruits, grains, vegetables and fish. An experienced parasitologist, using microscopy, may also be able to determine the parasite composition of a fecal sample that can then be compared to the parasite composition of a fecal sample obtained from a suspect. The presence of enterococci bacteria in the sample is also strong presumptive evidence for the presence of fecal matter. Vaginal Secretions The test for vaginal secretions is presumptive in nature as well. Vaginal epithelial cells contain a high amount of glycogen. Other cells in the body also contain glycogen such as oral and anal epithelial cells. By exposing a smear of the questioned vaginal secretions to iodine vapor, the glycogenated cells will stain a chocolate brown color. The non-glycogenated cells will be a yellow gold color. This test is called Lugol’s test. This test is highly subjective since a positive result is called when the majority of cells stain chocolate brown, however other types of cells may contain this high amount of glycogen. Because of this, the Lugol’s test is not widely used for casework purposes. Module 4: Laboratory Analysis of Biological Evidence (Semen and Other Biological Evidence) Page 6 Hair Hair The types of testing available for hair evidence are either comparative or biological. For comparative hair testing, physical and chemical characteristics can be determined and compared to a known hair sample from an individual. This type of analysis is typically performed by a trace examiner. Hair can also contain a root which may have suitable amounts of DNA for characterization or the shaft may