Study Guide DNA and Serology PDF

**[Core classes to be tested on:]** Biological Evidence and Serology Forensic Analysis of DNA Blood Spatter and Distribution Laboratory QAQC **\*\*Please remember that this study guide is not all inclusive; this is just to get you started in case you need some guidance. Please review the core c...

**[Core classes to be tested on:]** Biological Evidence and Serology Forensic Analysis of DNA Blood Spatter and Distribution Laboratory QAQC **\*\*Please remember that this study guide is not all inclusive; this is just to get you started in case you need some guidance. Please review the core classes listed above in detail.** **History** -- basic important people and general time frame of when discoveries were made - People - 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; "every contact leaves a trace - Karl Landsteiner- (1901), 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 - Ludwig Teichman- (1853) The first confirmatory test for hemoglobin - Paul Theodor- (1901), A method for distinguishing between animal and human protein first publish in scientific literature - Leones Lattes- (1915), Methods were developed for grouping dried blood stains - Landsteiner and Weiner- (1915) Rhesus blood factors were identified - A. Ochterlony (1949)- the ochterlony species identification test developed - Stuart Kind- (1960), Tests were developed for species identification using mixed agglutination methods - Brian Culliford- (1964) Polymorphic variants of the phosphoglumutase (PGM) enzyme were identified - Alex Jefferies- (1984), Seminal work in DNA fingerprinting was first published in scientific literature; developed restriction fragment length polymorphism - Kary Banks Mullis- (1986), The polymerase chain reaction was invented - Herbert Leon MacDonell- (1971), outlined several rules regarding blood spatter evidence; The average terminal velocity speed for a falling blood drop is 25.1 feet/second, reached in a max falling distance of 10-25 feet - Eduard Piotrowski- (1985) First to conduct official studies on blood stain patterns; with work published - Alexandre Lacassagne- developed study of gunshot wounds - Victor Balthazard (1939)- conducted work on blood spattered patterns - Time frames and specific dates - 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 - 1923- DC rejected scientific validty of a polygraph test - 1955- State of Ohio v Samuel Sheppard- blood spatter evidence indicated third person present at scene; Sheppard's DNA was left out of DNA testing - 1963- Brady v Maryland case: Mr Brady, along with two co-defendants was charged with murder committed during a robbery. One of the co-defendants confessed to the murder, however this confession statement was withheld from other statements turned over to the defense. This action was considered to be suppression of evidence that would have been favorable to the defense of Mr Brady. - 1970s- Further research determines that polymorphic variants exist for a number of serum proteins and blood enzymes - 1972- Giglio v US: John Giglio was accused of forgery and evidence primarily consisted of testimony of his co- conspirator. Mr Giglio\'s co-conspirator was offered immunity in exchange for his testimony, however that information was not disclosed to Mr Giglio\'s attorney nor was it disclosed to the jury. Similarly to Brady, the Supreme Court decided that the information withheld would have been important for the defendant and jury to know. - 1986- DNA profiling applied to the forensic case and the identification of Colin Pitchfork - 1988- FBI begins using DNA analysis in casework - 1989- HLA DQα was developed - 1992- The National Research Council publishes its first report (NRC I) DNA Technology in Forensic Science - 1993- The first STR is available - 1996- The national Research Council publishes its second report (NRC II) The Evaluation of DNA Evidence - 1998- FBI establishes CODIS DNA database; DNA Advisory Board (DAB) establishes quality assurance standards for forensic DNA laboratories - 2001- Melendez-Diaz v Massachusetts: In Melendez-Diaz vs Massachusetts, Mr Melendez-Diaz was charged with distribution of cocaine. The state presented the evidence as well as certified laboratory results. Citing Crawford v. Washington, Melendez-Diaz argued that the confrontation clause required expert witnesses to testify in person. Upon conviction, Mr Melendez-Diaz appealed stating that his 6th amendment right to face his accuser was violated. The Superior Court found that the practice of presenting a certified laboratory report was in fact in violation of a defendant\'s 6th amendment rights. This decision, of course, caused an increase in the need for Forensic Scientists to appear in person **Basic tenets of forensic science --** understand basic principles, value of evidence and types of evidence and their significance. - **The basic tenants of forensic science is based on observations by Edmond Locard: When two objects came into contact that was a bi-directional transfer of material between two objects (Locard Exchange Principle)** - **Types of evidence routinely recovered in serology/biology cases** - **Blood** - **Semen** - **Hair** - **Fibers** - **Evidice routine found with a vehicle scene** - **Hair** - **Clothing** - **Paint** - **Glass** - **Blood** - **Tissue** - **Whole Blood** - **Used as reference for DNA analysis** - **Obtained from victim or suspect** - **Collected in a sterile tube containing EDTA (which inhibits the activity of enzymes that degrade DNA)** - **Short term storage: refrigerated** - **Long term storage: freezer** - **Bloodstains and Mixed stains** - **Wet items must be dry before packaging to prevent contamination and cross transfer of staining** - **Short term storage: RT, humidity-controlled room away from direct light** - **Long term storage: dried and stored in low temp frost free freezer** - **Hairs** - **12-24 hairs is needed** - **Short term storage: humidity-controlled room; RT** - **Long term storage: frost free freezer** - **Intact roots for STR** - **Hair shaft for mtDNA** - **Swabs** - **Obtained from wet biological materials** - **Sterile swabs must be dried prior to packaging in a vial with a small aliquot of sterile TE buffer for short term storage** - **Blood droplets** - **Vaginal cavity** - **Rectal cavity** - **Buccal cavity** - **Obtained from inanimate objects** - **Sterile swabs must be moistened to collect a dry sample; then dried prior to packaging in a vial with a small aliquot of sterile TE buffer for short storage** - **Cigarettes** - **Envelopes** - **Bottles** - **Bone, Teeth, and Tissue** - **Section of 1cm\^2 is needed** - **Once collected, freeze and transport to lab on ice and kept frozen until analysis** - **Tissue samples, smears and slides** - **From stored biopsies or tissues from surgical procedures** - **Storage: RT indefinitely prior to DNA analysis** - **Semen and Sperm** - **Collected from vaginal swab** - **May contain epithelial cells** - **Sperm cells differentiated via extraction methods** - **Pure spermatozoa from condom: processed like bloodstains and mixed stains** - **Storage** - **Stored frozen prior to analysis** - **Avoid multiple thaw cycles** - **Urine** - **Once concentrated, it contains a sufficient number of epithelial cells to generate a DNA profile** - **Minimum volume of 10 mL** - **Storage** - **Frozen and refrigerated for short time prior to analysis** - **Concentrated samples can be obtained from stan or swab of known source** - **Fingernail Clippings** - **Collected from victim or crime scene** - **Swabbed using sterile moisten swab** - **Place in a container for storage or placed in a vial containing an aliquot of 1x TE buffer for short term storage** - **Long term: same as stains** **Crime Scene Investigation -- all aspects: securing the scene, contamination, searching and collection of evidence, preservation, packaging and storing of evidence. Types of searches, recording the scene and scene walkthrough, first responding officer.** =============================================================================================================================================================================================================================================================== - Securing the scene - Done by initial responding officer - Identifies anyone present at the scene - Prevents alteration or destruction of scene - Boundaries of scene - Where crime occurred - Potential points of entry and exits of suspects and witnesses - Places where the victim or evidence may have been moved - Processing scene - Survey - Photo/video documentation - Mapping - Recording and documentation of location of evidence - Searching for fingerprints or other physical evidence - Recording the scene - Documentation - Sketches, notes, photos, and videos - Contamination - Controlled by limiting access to those directly involved in scene processing - To minimize cross contamination: specific areas should be designated for trash and equipment and single-use materials should be used for the collection of biological materials ad other trace evidence - Walk through path to be establish as to no contaminate the scene - Search patterns - Strip or lane search - Grid search - Spiral search - Used in underwater crime scenes; start at key evidence and extend outward in a spiral pattern - Quadrant search - Final walk through is conducted to ensure all evidence has been collected **Types of samples**: be able to discuss all different types of biological samples that can be collected, how they are collected, packaged and stored. SEE EVIDENCE Human Remains and Identification -- means of identifying time of death and the value of markers of death. Be able to describe and give examples. Measures taken with outdoor scenes, human remains and indoor scene, environmental factors ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ - Notes taken when human remains are found - Position of body - Description and state of clothing (removed, stained, tangled, etc.) - Description of location, intensity, size, dir4ection of flow of staining - Type of stain (blood-like, semen, dirt, vomit, feces, urine or other fluid) - Any visible damage 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) - Outdoor corpse - May indicate partially decomposed or damaged by insects or animals or crucial evidence lost at original location - Evidence may have been placed around the crime scene - Estimating time of death - Changes in the eyes like dulling of cornea or appearance of a film - Body temp cooling that can be related to the outdoor conditions - The temp 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 - Cooling of the body after death when the ambient temp is cooler than the body temperature - A nude body will cook at 1.5 degrees C per hour when ambient temp is 18-20 degrees C - A body reaches ambient temp at 18-20 hours - Rigor Mortis - Rigidity that appears 6-12 hours after death due to biochemical changes in the muscle tissue - Degree and extent of rigidity is used to estimate time of death - Remains for 2-3 days then disappears - Livor Mortis - When blood circulation stops blood pools in the lower extremities of the body due to effects of gravity, causing a purplish appearance - Location of lividity can be used to determine if body has been moved - Putrefaction - Breakdown of the body due to autolysis and bacterial decomp - This coverts tissues of the body into liquids and gas that cause discoloration, bloating, and swelling - Fluid filled blisters appear on the skin - Internal gases force stomach contents out of the mouth, nose, and anus - w/in 1 to 3 years all soft tissues are gone leaving only bones - Mummification or formation of adipocere - Preservation causing a waxy covering that develops in 6-8 weeks as body fats are hydrolyzed **Evidence Handling** Documentation - Chain of custody Reference and standards (controls) SEE EVIDENCE Appropriate packaging of all different sample types - Most common packaging - Clean, unused paper containers like packets, envelopes, and bags - White butcher paper may be used to wrap evidence - Requirements of all packaged evidence - Seal w/ tamper-proof tape (never stapled) - Seal initialed - Packaged labeled with case number, item number, and location of collection - Packing of dried stains - Paper bag or envelope - Ways to remove stains from larger items for evidence collection - Cutting out a manageable section - Removing stains using cotton swabs - Scaping off dried stains - Utilizing tape lifts - Packing of wet stains - 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. - storage time must be minimal (less than 2 hours) to avoi8d bacterial degradation and sample putrefaction - once at lab the item must be hung to dry then placed in paper once dry - each container must have collecting persons initials; date and time sample was collected; complete description of the evidence and location found; and investigating agency's name and case number **Crime Scene Safety** **Routes of Exposure** - Inhalation - Intake of airborne pathogens (anthrax) and volatile chemicals - Skin contact - Frequent route of entry into the body - Chemicals can cause local irritation, redness, swelling, burning, or damage to the contact of tissues - Severity depends on type of chemical, concentration, and duration of exposure. - Systemic effects - Dizziness, nausea, organ damage, shock, and unconsciousness - Ingestion - Can damage mouth, throat, and digestive tract - Absorbed through the body via stomach and intestines - Minimization of ingestion can be accomplished by regular hand washing and abolishing the consumption or presence of food and drink in contamination prone areas - Injection - Needle sticks and mechanical injuries from contaminated glass, metal, or other sharp objects **Universal Precautions** - Definition: fundamental mechanism considered necessary to main the BBP standard and are primary mechanisms for infection control - BBP standard - Directs employees to treat all blood, body fluids, or other potentially infectious materials as if they are infected with HBV, HCV, and HIV - Protective measures to take to avoid direct contact with infections materials - Barrier protection- gloves, coveralls, shoe covers - Eye and face protection - Place contaminated sharps in closable, leak proof, puncture resistant containers when transported or discarded and label w/ biohazard warning - Prohibit eating, drinking, or applying cosmetics where human blood, body fluids, or other potentially infectious materials are present - Well-fitting respirators - Instruments used are to be cleaned with soln of bleach diluted 1:10, 70% OH, or other disinfected - Biohazardous waste storage and disposal is regulated - Any waste capable of producing an infectious disease - Cadaver waste, human blood, body fluids, and tissues are waste - Universal Precautions - Assume all samples are infections chemicals - Maintain max protection at all times while minimizing exposure potential - Liquid blood can carry HIV, Hep A/B/C - Use appropriate PPE Personal Protective Equipment - Hand protection - Gloves - Checked for holes and tears consistently - Eye protection - Safety glasses and goggles; Face shields during possible splashing - Foot protection - Shoes that completely cover and protect the food - Possible shoe covers when needed - Respiratory Protection - Bombings and clandestine labs can produce noxious fumes making respiratory protection necessary - Chemical Safety - MSDS - Material safety data sheets - Provided by manufacturer or supplier - Notes all hazards and recommended PPE, handling, and storage -- -- -- -- -- -- Chain of Custody -- what is it; why is it important; what are its limitations. ------------------------------------------------------------------------------ - Required to maintain COC - A description of the evidence and its container - The specific recovery location of the evidence - Case numbers - The date and time 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 evidence - Reason for COC - Have the best chance of maintaining the integrity of the evidence - You cannot testify to what happened to evidence while in someone else custody, each person handling evidence follows policies and procedure developed to keep the evidence secure and free of contamination **Searching and note taking in the laboratory: how does the processing occur? Know the basics of the examination procedure followed by forensic biologists in the lab. Understand the process of what happens from the time a sample is collected in the field and ultimately stored.** - **Step 1** - **Analyst selects an isolated area to examine evidence** - **Step 2** - **Wearing the appropriate PPE, bench area covered with clean paper prior to opening evidence** - **Paper is changed after every item** - **Step 3** - **Packages of evidence are checked against evidence list in case file** - **Where necessary, evidence items are divided into those obtained from those below and placed in complete separate areas or rooms:** - **The subject** - **The victim** - **The scene** - **Step 4** - **The item selected to examine is opened in a manner that preserves the original seal and signature** - **Step 5** - **Notes are made at each step, item sketched and searched** - **debris and potential evidence recovered** - **recorded and packaged** - **fibers and hairs are mounted on glass slides** - **other trace evidence is packaged and submitted for analysis** - **Step 6** - **Appearance and location of stains or damaged are noted** - **Location of any trace evidence is recovered is noted** - **Notes can reflect characterization of the item so that it can be easy to identify in court** - **Step 7** - **Presumptive tests are carried out on stains** - **If stains on clothing are negative, the rest of the item needs to be swabbed and testing in case the stain is not visible to naked eye** - **Step 8** - **Areas of a stain tested positive are selected for confirmatory tests and DNA analysis** - **Genetic difference from suspect stain and reference sample allows the source to be positively eliminated** - **Step 9** - **Once examination is complete, the item of evidence is wrapped in the exam paper that it is opened on so that no trace evidence is discarded** **Presumptive and confirmatory tests** -- what is the difference, demonstrate you understand the difference, give examples, which are done in the field and which are done at the lab. - Chemical on scene tests - Sensitive but not specific tests for body fluids that depend on detection of constituent chemicals - Presumptive blood tests - Rapid, chemical tests that may be used to locate and differentiate between blood and other similarly colored stains - Highly sensitive, easy to perform, and allow traces of blood to be located where they may not be easily seen - Most rely on reactions associated peroxidase activity of hemoglobin - Most depend on oxidation of colorless reduced chemical indicators - Where the reaction with blood results in a color change in reagent - Usual test sequence - Add drop of reagent to stain - Add hydrogen peroxide - Rapid color change is a positive result - Anything longer than 20 to 30 seconds is negative - Common color tests for blood - Benzidine - Indicator color: BLUE - High sensitivity - Tetra methyl benzidine - Indicator color: BLUE - Medium sensitivity - O-Tolidine - Indicator color: BLUE - Medium sensitivity - Derivative of benzidine and has health risks - Can produce false positive with gun metals - Phenolphthalein or Kastle Meyer test - Indicator color: PINK - Low sensitivity - Most common - Can be performed as a 3-step test - Step 1: rub stain with filter paper then addition of a few drops of ethanol to increase sensitivity - Step 2: addition of phenolphthalein (this may cause a color change if an oxidizing agent like plant peroxidases are present) - Step 3: addition of hydrogen peroxide which in the presence of hemoglobin oxidizes colorless reduced phenolphthalein to pink phenolphthalein. - Leucomalachite green - Indicator color: GREEN - Low sensitivity - Most common - Reagent: sodium perborate, leuchomalachite green, glacial acetic acid and water - Procedure - Few drops of reagent added to filter paper swipe of stain - Add H2O2 - Rapid color change indicates blood - Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) - Indicator: FLUORESCENCE - Very high sensitivity - Hemastix - Used by responding officers at crime scenes or hospitals - Reagent stick is touched to stain in question and drop of sterile water is added - Green color indicates blood - Can give false positive to gun powder residues - Can be discouraged because then the sample goes through multiple tests decreasing the likelihood of obtaining a good DNA profile - In lab confirmatory tests - Confirms the presents of blood - Microchemical tests - Confirms the identity of a positive presumptive test for blood - Takayama or hemochromogen test - Ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin - Once a reaction occurs, the crystal is observed under a microscope - Teichman reaction - Works the same as Takayama - Of the two, the Takayama is preferred because it is easier and the crystals tend to form with a range of stain types - Can be disrupted by dirt and other particulate matter present in the sample - Microscopy - Used to verify the presence of blood cells in a suspected blood sample - Dried stains - Cells must be reconstituted, re-dried, fixed and stained for microscopy for the identification of characteristic morphological features - Depends on the level of expertise of the analyst and their ability to find intact cells within the stain - Integrity can be affected by the age of the stain, the condition of the stain and the nature of the surface the stain is on - Spectroscopy - Distinguishes between hemoglobin and hemoglobin derivatives - Reliable technique for older stains that have tested negative using color or microcrystal tests - All hemoglobin derivatives exhibit a strong absorbance at 400-425 nm (soret band) - Hematrace - ABAcard hematrace tests - Detects the presence of human hemoglobin in a sample - Based on antibody-antigen reaction between the sample and the antibodies in the test card - Shown to react positively to ferret blood Lab analysis ------------ All, or the majority, of a forensic analysts work takes place in the laboratory not in the field. You need to demonstrate your understanding of this; know the difference between locating a stain/searching techniques and presumptive tests in the laboratory (not at a crime scene) see above and below **Testing** Know examples of presumptive and confirmatory tests whether they are conducted at the lab or in the field. Be able to describe the steps of a test as well as the detailed process of evidence examination and analysis. Know the difference between the terms: examination and analysis. - Examining the area for blood (presumptive testing in the field) vs. analysis of the blood (confirmatory testing in the lab) **Be able to describe in detail, with examples:** Common Color Tests for Blood (presumptive) see above Microchemical confirmatory tests for blood see above Spectroscopy and Hematrace see above Semen -- searching techniques, blooting - Searching Techniques - Visual and Tactile examination - Semen stains on evidence can sometimes be visible to the naked eye - White stain on dark surfaces and yellow on white surfaces or is old - Has a grainy texture - ALS may be needed to highlight stains. - Crusty touch test - Gently running gloved fingers across the surface to locate the crusty stain - Visual should always be performed first and areas suspected be marked w/ a wax pencil. - ALS - Useful w/ bedding and vehicles - Semen has fluorescing properties due to bacteria (pseudomonas fluorescens) and flavins - Visualized using 400-500 nm wavelengths and orange safety goggles - Must be done in a dark room - Since other body fluids can be seen with ALS, this is not a test for semen alone - Blotting - Useful on clothing and bedding - Uses mapping process - Damp blotting paper is applied to the item following a grid pattern and labeled to indicate location on item - Papers pressed against item then removed to fume hood to be sprayed with color tests reagent - Determines whether acid phosphatase is present and to determine the distribution of staining on an item - Con: the damp blotting paper can cause a high risk of transferring the original stain to new areas where the layers of fabric overlay - This is avoided by using polythene between the upper layer of fabric being tested - Item must be fully dried before repackaging - Each stain is mapped with wax pencil then sent for confirmatory testing - Take small clipping of each AP positive area and place into a small tube with a few mL of distilled water in prep for microscopy Semen -- presumptive tests: acid phosphatase spot test, BCIP test - Presumptive testing - Semen contains high concentrations of acid phosphates which is secreted by prostate gland - It is responsible for cleaving phosphate from phosphorylcholine yielding choline for cell membrane composition and repair - Presumptive testing for semen is based on hydrolysis of phosphate esters and the detection of liberated organic colored complex - Acid phosphatase spot test - AP testing is the most common - If acid phosphatase is present the sample will react with sodium alpha-napthyl phosphate to form naphthol - Naphthol when combined with Fast Blue B forms a purple azo product - 1 step process - Sodium alpha-naphthyl phosphate and Fast Blue B are combined into one reagent then applied to stain in question - A change to purple coloring means possible presence of acid phosphatase - 2 step process - Sodium alpha phosphate and fast Blue B are used as separate reagents where 1 drop of reagent 1 is applied then one drop of reagent B - BCIP Test - 5-bromo-4-chloro-3-indolyphosphate - Performed by taking a swab of the stain in question and adding to a tube filled with BCIP soln - If AP present it will hydrolyze the BCIP creating a blue color to change w/in 15 min - Both have false positives - To vaginal acid phosphate that is typically elevated in prepubescent females - To blood from erythrocytic acid phosphatase and seminal acid phosphatase that is elevated in males with prostate cancer - To male urine with AP present - To plant matter, feminine hygiene products, and spermicides - Negative testing - Results in no further testing - Sexual assault kits should be confirmed regardless of presumptive testing - Sperm last longer in body cavities - Semen Confirmatory tests - Microscopy - Most reliable techniques - Histological staining - Light microscopy - Preparation - Cut a tiny piece of a swab from the SA kit and place in micro centrifuge tube with DI water - Tube is agitated and centrifuged creating a pellet at bottom - Pellet cells are applied to glass slide and heat fixed - Stain applied - Christmas treet staining: Includes nuclear fast red to stain heads red and picric acid-indigocarmine (PIC) to stain tail green - Can also be Hematoxylin and Eosin stain which makes head purple and tail pink - Sperm pains: fluorescent dye mixed with antibodies to bind to sperm head and tail antigen - Selective only for sperm cells and will not stain any other debris or cells - Can be used with laser micro-dissction microscoptes - These find sperm cells on a slide then dissect only but the sperm with a laser to isolate for DNA testing - Positive results - When any number of intact heads and tails are observed on the slide - Presence of one or more heads with no visible tails depending on labs - Intact vs non-intact - Spermatozoon with or without a tail is still a spermatozoon - Microscopic exam - Looking for differential staining for the presence of acrosomal cap on the head or tail, morphology in general of the sperm, and its size - Sperm is bigger than bacteria and yeast but smaller than epithelial cells - Dog sperm is 3x bigger - Positive presumptive test but negative confirmatory test possibilities - Oligospermic - Low sperm count - Aspermic - No sperm present in seminal fluid - Further testing will need to be done to determine the present of p30 or prostate specific antigen (PSA) - Only found in high conc in human semen - PSA/P30 - What is it? - Secreted by prostate gland and it liquefies semen and aids in dissolving the cervical mucus cap to aid in sperm entry - Detection - By precipitin reaction with antiserum using Ochterlony process - Quantitative immunological testing using ELISA - PSA survival with this technique has shown to be eliminated within 234-27 hours after intercourse - PSA survival post coital vagina - 6 hours to 3 days - Most common testing - Detect the presence of PSC based on an antibody-antigen reaction between the sample sand antibodies on a test card (much like Hematrace for hemaglobin) - ABAcard - Seratec PSA semiquant - Works the same as ABA but has a semi quantitative4 internal control that aids in detecting semen concentration - Can sometimes be a confirmatory test - Important in cases with vasectomized or aspermic individual to confirm semen when sperm is not present and cannot be confirmed with microscopy - Can be present in blood of males with prostatic cancer and in male urine - Not as high of conc and will not show up on PSA testing - TSI interval - Time since intercourse - Parameter used to determine whether recovered semen is due to the reported assault rather than from a consensual sexual act that occurred before assault - Why orifice swabs are not taken - Motile sperm is not there - Sperm can survive in the vagina 3 hours after ejaculation (per hospital data) - Survial time of sperm - 1-8 hr depending on conditons - Motile sperm survive longer in cervix (sever hours or days) - Intact spermatozoa persist in vagina up to 26 hours and heads 3-7 days - Dependents of sperm count - Penetration - Complete ejaculation - Variations in sperm count - Victim's activity following incident - Sperm survival in rectum - 6-65 hrs - Sperm survival in oral cavity - 6 hours due to saliva and drinking - Reference samples to be obtained - Suspect if known - Elimination reference samples of consensual partners **Other types of analysis**: Saliva, urine, feces, vaginal secretions, touch DNA - Saliva - Detection - Difficult to see with naked eye - ALS may fluoresce at a lesser extent than other body fluids - Methods: use of the location of the salivary enzymes alpha amylase - Depends on ability of amylase enzyme to split water-insoluble amylose bound to dye to soluble saccharide products that result in color change - False positives - Amylase in low conc can be found in vaginal fluids, breast milk, swear, blood serum, semen, and fecal material - Screening - Amylase mapping - Similar to seminal fluid searching - Large sheets of blotting paper stained with test reagent and moistened then present to area - Most common reagent - Phadebas - Tablets that consist of blue dye cross-linked to starch (beta-1-3 glucose bond) - Positive reaction: - The starch is digested releasing the dye into solution resulting in a blue color - Used quantitatively - More sensitive and specific than mapping - Amylase substrate is cross-linked to starch and made into a tablet - Amylase present in sample hydrolyses the polymer into a blue starch than can be measured calorimetrically at 620 nm. - An absorbance value of 0.3 absorbance units and greater is a positive reaction - Procion Red MX2b amylopectin (Lyosine red) - The reagent is pink and if the paper comes in contact with saliva the area with saliva turns white - Positive tests infer presence of saliva but do not confirm - Radial diffusion testing - False positives - Mixed fluid stains - Procedure - Agrose gel containing 1% starch with holes punched out is used to place extract of unknown sample. The gel will diffuse and produce a clear area after staining with iodine soln - Size of clear area is directly proportional to amount of amylase in sample - General swabbing - Swabs of an item where saliva may be indicated or taking representative cuttings - From bite marks - Wiping area with moisten cotton swab - Swab air dried and then testing - From clothing - General swab of area where saliva may be indicate - Cuttings from these areas - Uses for saliva testing - Burglary mask found - Amylase mapping to pinpoint saliva inside mask - The location of saliva stain may contain a high enough concentration of epithelial cells to develop a DNA profile - Reasons for no amylase testing in labs - Saliva is difficult to isolate - People have varying levels of amylase activity - One person's saliva may react while the other produces a false negative - Saliva in SA - Reasons for amylase testing - Saliva is used as lubricant for juvenile SA and may be detected on underwear - Oral assault/victim was unconscious - Protocols for before amylase testing - Sweeps or removals of hair, paint chips, soil, gunshot residue, or fibers BEFORE testing - Urine - Basic info - Net filtrate from kidneys - High concentrations of urea and creatinine - Yellow color from pigment urochrome - Odor due to bacterial breakdown of urea - ASL can be used to find urine stains - Fluoresces faint blue-white - Chemical testing - Uses colorimetric detection of ammonia produced from urea by urease - Presumptive test - Urea litmus paper test indirectly indicates the presence of urea by reacting the sample to urease which generates the ammonia - Urea-nitrogen test - Ammonia is indicated by the production of a deep blue color reaction - Urea + H20 -\> CO2 +2NH3 - Jaffe test - Detects creatinine that is eliminated through the kidneys - Forms a red compound with the addition of picric acid - No confirmatory testing - Microscopy - Relies on the fact the urine contains several solid materials with crystalline structures - Feces - Basic info - Unabsorbed residues from gut - Include food, gut wall debris, digestive secretions, and bile pigment breakdown like urobilinogen - Urobilinogen is a bile pigment excreted in feces and reactions with this compound form the basis of presumptive tests for feces - Odor due to skatole - Chemical tests: Edelman test - Urobilinogen is oxidized to a pink-red produce with mercuric chloride; using 10% soln of mercuric chloride in amyl alcohol as reagent - The suspended sample is centrifuged, and the supernatant is treated with zinc chloride - Microscopy - One of the oldest methods - A sample of feces will contain the undigested residues of food digest within the last 12-24 hours - The presence of enterococci bacteria in a sample is strong presumptive evidence for the presence of fecal matter - Vaginal Secretion - Presumptive - Vaginal cells contain a high amount of glycogen - So do oral and anal epithelial cells - Lugol's test - A smear of questioned vaginal secretion is exposed to iodine vapor - Glycogenated cells will stain a chocolate brown color - Non-glycogenated cells will be yellow gold color - Highly subjective - A positive result is called when the majority of the cells are brown - Not widely used for casework purposes - Touch DNA - Wear Areas and Touch - Wear areas are when pieces of evidence like clothing aid in determining the wearing of the item - These areas contain sweat or skin cells from friction of wearing the item - Collected by swabbing the area or taking a cutting - What is touch DNA - DNA left behind from a person touching or handling an item - Ex. Swabs from steering wheels, gun handles, door handles, shell casing - Factors in obtaining probative results from touch DNA - Shedders vs non-shedders/sweating - Some people shed more skin than average (shedders) - Some people shed less skin than average (non-shedders) - If sweating when touching an item, there may be more DNA transferred from the touch since the sweat acts as a vehicle to transfer skin cells - Surfaces being touched - Rough textures cause more friction - Items with larger surface area - Ex. Shell casings have much less chance of producing DNA resultants than a steering wheel - Length of time touched - Repeatedly touched item will have higher amounts of touch DNA - How many people have touched it - Door handles or steering wheels are less likely to produce probative DNA results due to complicated mixtures - Presence of other sources of DNA - Item w/ blood or other body fluid contains high concentrations of DNA and cannot be tested - Factors that may affect sample collection of touch DNA - Surface type - Rough/textured: one or two moist swabs - Careful not to shred the swab - Follow up with dry swab if surface is wet after - Size of surface - May require more than one swab - Keep swabs to minimum and never use gauze pads to take a swabbing b/c it spreads a minimal ample over large surface of gauze - Cleanliness of surface - Wet swab followed by dry swab - Latent print request on same surface - Latent print processing can dilute the existing sample - Open communication with latent print examiner will help preserve evidence - If you can do one or the other, determine how well you may get a touch DNA sample vs a latent print - Presence of other DNA - Blood or other body fluid on a surface should not be tested for touch DNA unless the fluid is elsewhere - if you swab for both - swab areas separately - do not allow your swabs come into contact with each other and package separately Be able to describe in detail, with examples, all step-by-step processes used in the lab analysis of biological fluids/samples - Electrophoresis - Separation technique that utilizes the face than an electrically charged molecule placed in an electric field will move towards the electrode with opposite charge - \+ molecules move to cathode - \- molecules move to anode - Due to magnitude of each charge, each molecule separates and migrates at different rates - Used forensically to determine biological markers in suspect stains for personal identification and in the early process of DAN sequencing DNA analysis for identification - Used today for protein analysis - Net charge depends on amino acid composition of that protein - Can be used to detect subtle variations in protein charge from AA substitution - Visualization - Hemoglobin is naturally colored and can be visualized once separated - Proteins, antibodies, and enzymes are non-specifically stained using: - Ponceau red - Amido black - Coomassie blue - Species Typing - Some labs do ABO testing to skip - Helps in mass disasters - Identifying blood proteins that are specific to each animal species - Rely on interactions of antibodies and antigens - Antibody- protein produced by the body's B cells in response to the presence of foreign body, or antigen - Primary immune response and function to prevent or minimize disease by attaching themselves to a foreign antigen - Techniques - How it works: antigen-antibody principle used to produce a precipitin type reaction between the specific antigens present in an extract of a test stain and antibodies present in the reaction antiserum - Immunoprecipitation - Antibodies combine with an antigenic determinant and a visible precipitate forms - Electrophoresis - Specificity - Capability of an antibody to recognize only one particular antigen - Most important parameters in species identification - Cross reactivity - First way: the antiserum is not tissue specific and will give reactions with other or closely related species - Second way: Not species specific and will give reactions with other closely related species (sheep w/ goat) - Occurs b/c relationships between species are reflected in similarities in protein sequences - Ouchterlony Double Diffusion - Most popular species determination test - Works on basic principles of immunodiffusion and subsequent precipitation - Procedure - Adding antigen and antibody to several separate small wells in an agarose gel - Antigen and antibody radially diffuse from each well towards each other, forming precipitin lines where antigen-antibody reactions occur - The band formation provides info on identity, partial identity, or non-identity - DNA testing - Instead of identifying presence of human protein they identity presence of human DNA - Uses oligonucleotide probes that hybridizes to human DNA that has been immobilized on a nylon membrane. - Proble D17Z2 is a 40 base oligonucleotide that is complementary to primate specific alpha satellite DNA sequence on chrome 17 - Alpha satellite DNA is composed of highly repetitive sequence elements found near human chromosome centromere and is chromosome specific - Results - DNA tests are less prone to false positive results than immunological tests due to lack of cross reactions - Results under limit of detection should be noted as inconclusive - If semen stains are aspermic then DNA results depend on amount of non-spermatozoa cells present and the size of the sample - DNA samples will give negative result but immunological will give positive - An immunological sample that is negative for human DNA can be tested with anti-animal serum Serology -------- ABO Blood Grouping ------------------ - Red cell antigens - Isoantigents -found In in certain individuals that are immunogenic in some other members of the same species. - Types, their serum, and their ABO typing - "A" Red cell Ag has serum "B" Ab and the ABO type of A - "B" Red cell Ag has serum "A" Ab and the ABO type of B - "A and B" Red cell Ag has no serum and the ABO type AB - No Red cell Ag has serum "A" and "B" and the ABO type of O - Phenotypes and Genotypes - AB phenotype is AB genotype - A phenotype is AA or AO genotype - B phenotype is BB or BO genotype - O phenotype is OO genotype - Structural differences of the ABO red cell antigens - H antigen is the precursor to A and B antigens - The B gene produces a transferase that adds D-galactose to the structure of the H antigen - The A gene produces a transferase that adds N-acetyl-D-galactosamine to the terminal D-galactose of the H antigen - The O gene doesn't produce any enzymatic activity, so the H antigen remains unchanged Secretor Status -- what is it; how does it help? ------------------------------------------------ - Secretor status - People can express their ABO antigens in a water-soluble found at high concentrations in body fluids like saliva, semen, sweat, tears, and serum. - Group O people secrete H antigen - Group A people secrete A and H antigen - Group B people secrete B and H antigens - The secretion process is genetically controlled and those who possess the secretor gene are called secretors ABO stain grouping - Stain grouping - More complex than other samples because the red cells in blood stains are ruptured or lysed - Also, difficult due to stain or sample contamination; limited amounts of sample; exposure to harsh environments, and substrate interference like when exposed to strong detergents. Blood Enzymes and Proteins **- examples, tissue distribution and storage** - Blood Enzymes and Proteins - Genetically controlled traits - Polymorphic enzymes - Enzymes which have slightly different structural forms, usually due to minor differences in the AA comp but have similar functional properties - Previously used to help determine origin of biological stains and fluids - RBCs, serum, and other body fluids contain numerous polymorphic enzymes that provide biochemical markers that can be determined using gel electrophoresis - Differences in the enzyme patterns combined with traditional ABO blood grouping can narrow down sample origin - The probability of a bloodstain having a specific combination of markers is the product of their distribution within the population - Haptoglobin - An alpha-2 globulin glycoprotein that readily forms a stable and irreversible complex with hemoglobin, and controls hemoglobin excretion from the body - The polymorphic nature is due to unequal gene crossover, resulting in production of allelic proteins with substantially different molecular weights - Commonly seen phenotypes - Hp1 (1-1), Hp2 (2-2), and Hp2-1 (2-1) - A valuable marker for serologists since it is very stable in dried stains - Phosphoglucomutase (PGM) - A phosphotransferase enzyme that catalyzes the reversible conversion of glucose-1-phosphate to glucose-6-phsophate during carb metabolism - Fond in plants and animal tissues and in micro-organisms - In humans, the enzyme is found in large quantities in blood and semen, and in smaller amounts in vaginal and cervical mucus - If stored properly, the enzyme remains active prolonged periods - Group Specific Component (Gc) - Vitamin D binding protein: a glycoprotein in the alpha-2 globulin fraction of serum proteins, produced by the liver - Presence may be decreased in the event of hepatic disease or pregnancy - Studies indicate that serum stains produce more reliable results than dried bloodstains - Studies with aged serum stains demonstrated that the GC protein in this sample type was significantly more stable than in aged bloodstains - Erythrocyte Acid Phosphatase (EAP) - 2 distinct types of acid phosphatases present in human tissues - Red cell or erythrocyte acid phosphatases have a low molecular weight and can be found in the cytoplasm of other tissues - Distribution of Genetic Markers in Tissue - Many genetic factors found in blood can be found in other body fluids and those found in body fluids may be found in blood - Protein markers may be present in sperm as well as seminal fluid - Genetic typing can be done after a male has been vasectomized - PGM can be found in vaginal secretions, but it degrades rapidly - Semen stains are easy to analyze since the dilution effect is less prominent - Saliva contains several polymorphic markers that are found almost exclusively in the saliva - only a few were found in blood or other bodily fluids - other tissues with polymorphic genetic markers - hair, tooth pulp, and inner ear fluid - Sample handling and storage - Protein markers deteriorate over time - This begins at the time a sample is shed and progressing as the sample is collected, stored, and processed. - Maintain integrity - The sample must be analyzed as quickly as possible - Appropriate handling and storage conditions - Wet stain degradation - Through the action of microbiological processes and endogenous enzymes - Drying of samples and storage in fridges slowed degradation processes - Non-enzymatic process like hydrolysis and disulfide exchange reactions can also modify proteins which affect electrophoresis patterns - Degradative reactions - Photo-oxidations, direct oxidations, and free radical reactions - Frozen or cold samples - Keep liquid samples cold or frozen - Frozen liquid sample provide active markers for moths to years depending on the marker - Dried samples - Freezing enhances marker stability Blood Spatter ------------- Hemodynamics, clot formation and bruising - Hemodynamics - Blood - A viscous liquid comprised of a liquid portion (plasma) and cellular components (RBC, WBC, and platelets) - Circulates through the vasculature of the body and function to deliver nutrients and oxygen to the cells of our body and to facilitate the removal of waste products - Carries glucose and hormones to our tissues and plays a critical role in the body's defenses by supporting antibodies and white blood cells that remove foreign particles form our systems - Oxygenated blood in arteries is bright red - Deoxygenated blood in veins is a darker purplish color - RBCs - Erythrocytes - Free of nuclei and play a key role in transportation of oxygen and carbon dioxide between the lungs and tissues - Platelets - Cell fragments that have broken off the parent cells (megakaryocytes) - Instrumental in coagulation - No nucleus - Life span of 10 days - WBC - Leukocytes; shorter life span than RBC - Defend the body against invaders and carried in the circulatory system to tissues - Types of WBC - Lymphocytes - Monocytes - Neutrophils- 6 hour half life - Basophils - Phagocytes - Neutrophils, monocytes, and macrophages - Can engulf foreign particles - Immunocytes - Lymphocytes - Responsible for specific immune responses - Granulocytes - Basophils, eosinophils and neutrophils - Contain cytoplasmic inclusions that give them a granular appearance - Blood cell production - Hematopoietic stem cells - Precursor to blood cells - Found in bone marrow - Hematopoiesis - Production of blood cells - Active bone marrow is pink/red and produces 25% RBC and 75% WBC - Inactive bone marrow is yellow - Controlled by cytokines (peptides and proteins produced by cells that affect growth and activities of other cells) - - - Heart - Functions as a hydraulic pump to push blood out through the dorsal aorta - Vessels can constrict (vasoconstriction) or widen (vasodilation) - Dorsal aorta - Main artery - Divides and branches into many smaller arteries that supply each region of the body with freshly oxygenated blood - Arteries - Muscular walls with strong elastic properties - Inner wall of epithelial cells lines the inside of the arterial wall which makes it smooth and allows the blood to flow unimpeded - The walls expand and contract as they fill with blood and push the fluid under high pressure throughout the vascular system - Arterial branches divide further into capillaries - The rhythmic pumping of the heart combined with the contractile forces of arteries circulates the blood efficiently - Capillary - Walls are on epithelium cell thick and are fragile - Blood cells flow through in a single file - Oxygen, carbon dioxide, nutrients, and waste products diffuse back and forth across the capillary wall to, and from, the surrounding tissues and organs - Deliver deoxygenated blood and waste products to veins for transport back to lungs for re-oxygenation and back to heart for recirculation - Veins - Less elastic than arterial system - Blood is transported back to the heart under less pressure - Valves positioned along each vein to prevent black flow and keeps it moving in one direction against the force of gravity - Angiogenesis - Formation of new blood cells - Occurs in children during growth and adults for wound healing, regrowth of uterine lining, and during endurance training - Controlled by angiogenic and antiangiogentic cytokines - Two main growth factors - Vascular endothelial growth factor - Fibroblast growth factor - Angiogenesis inhibitors - Angiostatin - Blood pressure - Highest in arteries - Decreases in pressure due to resistance of blood vessel to blood flow - Arterial breach - Pulsatile spurting - Venous breach - Oozing or flowing - Hypostasis - When irreversible circulatory arrest occurs, the absence of blood pressure, tissue turgor, underlying pressures and hydrostatic pressure results in blood pooling to lowest levels of cardiovascular system - i.e. a body on its back will have blood pooling in the back but, thighs, calves, and neck - early signs - patches that are small and lightly colored - later signs - patches join together and become darker - dark pink to blue - Clot formation and bruising - Homeostasis - Step 1: vasoconstriction - Injured vessel constricts decreasing blood flood - Step 2: platelet activation and plug formation - Step 3: coagulation - - Ways to protect itself when blood loss occurs - The body increases vasoconstriction and fluid retention, and platelets are activated - Platelet activation initiates a cascade clotting reactions at the site of an open wound - Platelets - Irregular shaped, colorless cells with sticky surfaces to gather at wound site and form a clot that will stop bleeding - Processes that include reactions with calcium, vitamin K, and protein fibrinogen - Once blood is exposed to the air, the platelets begin to break apart and react with fibrinogen to make fibrin threads - Fibrin threads form a mesh like web that traps blood cells and dries to form a clot or scab - Clot formation requires interaction of calcium and vitamin K, and insufficient or reduces amounts can result in abnormally slow, life threatening, clotting rates - Normal clot time - 3-15 min in healthy individuals - Bruising - Result of bleeding under the skin when superficial blood vessels are damage - The color of a bruise changes with age - Black/ blue are fresh - Green /yellow are older Physics of blood spatter: motion and directionality - blood motion and directionality can provide info on sequence of events, droplet directionality, and blood trail motion - Blood motion - A blood drop is held together by cohesive forces that produces a surface tension within and on the surface of the external surface of the drop - Surface tension- force that pulls the surface molecules of a liquid together that helps the liquid resist penetration - Surface tension of blood - Less than water - To create a blood spatter, the surface tension of a blood drop must be overcome from an applied external force - Shape of blood in air - A spheroidal shape due to the molecular cohesive forces acting upon the surface of the drop - Passive drop of blood - i.e. a blood dripping from a wound - occurs when the volume and mass of dop increase to the extent the gravitational pull on the drop overcomes the cohesive forces at the blood source - Amount of blood needed for a passive drop - Depends on the type of surface and the surface area from which the blood originated - Average measurements of blood drop - 0.05 ml which forms a spheroid about 4.5 mm in dia. - Velocity - The more viscous a fluid becomes, the slower it falls - Since blood is more viscous than water it has a high specific gravity than water - Viscosity and specific gravity of blood are responsible for the stability of exposed blood drops and their resistance to alteration and disruption - A blood drop will accelerate until forces opposing the drop equal the force of the gravitational pull, and the drop reaches terminal velocity - Average velocity for blood - 25.1 ft/sec reach in a max falling distance of 20-25 feet - Blood hitting a surface - Resulting spot has a diameter that varies with the volume of the drop, the texture of the surface it impacts, and the distance fallen - Uniform sized blood drops that are dropped from different heights ono a smooth hard surface produce increasing diameters - Dropped over range of 6 in. to 7 feet, the diameters range from 13-21 mm - At heights higher than 7 feet, diameter does not significantly change - Target surface - Smaller blood spot from a parent blood spot - The outer skin or surface tension of the parent spot must be broken - It will no break into small spots until it strikes a surface - Surface texture - Smooth, hard, nonporous - Creates less spatter - Rough textured surface - Creates more spatter - Have protuberances that rupture the surface of the drop, producing spatter and irregular stains with spiny and serrated edges - Four phases of impact - Phase 1: contact and collapse - Droplet hits target and begins to collapse from bottom upward - Phase 2: displacement - Collapsed sphere has the majority of the blood volume on the outside boundaries of the resulting stain - Dimples and short spines are formed - Phase 3: dispersion - Blood is forced to the boundary rim and dimples ride upward and opposite to direction of original momentum - Now unstable, the sphere breaks apart creating the satellite spatter - Phase 4: Retraction - Final phase - Results from effect of surface tension pulling the fluid back to a single form - Spines are pushed away from parent stain - Directionality - Geometric parameters are used to determine direction of flight before the drop hit a surface - Parameters derived from the stain edge characteristics of individual stains - Narrow end of an elongated blood stain points to direction of trace - Once direction is determined, a point of convergence that represents the relative location of the blood source is determined by joining straight lines drawn through the long axis of each stain - Area of origin - Determine by establishing the impact angles of individual blood stains and their trajectories - Point of convergence - Point where several stain patterns cross or intersect - Determined from overhead and from the side via string technique or specialized soft wear - Overhead examination - Deviations from standard paths - Intermediate obstacles or size limitation of room - Reverse path could be extensive depending on location of scene - Two stains may have an intersection point - May indicate the stains are the result of two separate/different events and point of convergence falsely identified - Stains with no common point of convergence can be considered two actions - The closer the stains are the more like they are related - More stains mean more confident in POC - Limitations - Cannot be used to estimate point of origin - POC established in one plain and assumed droplet's flight path is only established on one plane - Problematic when multiple events occur - Usefulness - When combined with other techniques - Useful for vertical target surfaces - Side view angles - Best method for determining POC - Uses angle of impact of the stain - Apply length/width ratio sine concept for angle of impact and use straight line impact angle formula to provide estimation of angle of impact - Limitations - Provides only estimation due to parabolic flight characteristics and oscillations in the flying droplet preventing the value from being absolute - Point of origin determination - Once measurements are obtained the POO can be determined - X axis is point of impact - Y axis is point of origin - (X,Y)0 is the POC - 3D estimations - "stringing" technique or computer assisted analysis - Stringing technique - Basic method - More of a visual aid in explaining the concept of POO in court - Process - Stains are selected and angles of impact determined - Strings or threads are taped to leading edge of stain - String extended back along reverse travel path at estimated angle - Point where strings intercept is the POO - Limitations - Time consuming - Fining appropriate attachments can be difficult - Uses props which result in clutter and limited movement around the scene - Strings cannot be placed along direct flight paths - Lasers are now used in the stringing technique - Computer software - Replace stringing - Used to determine POO and include parabolic flight characteristics in their analysis as well as air resistance, droplet volume and gravity - Stain measurements - Only main portion of stain should be measured - Avoid satellite spatter and spines - Length of stain is measured along major axis - Width of stain is measured across minor axis - Template technique can be used but has limitations: - Do not compensate for differences in blood volume that create wider or narrower stains - Can also be measured with drafting dividers or rulers - Angle of impact - 90° angle of impact has a circular blood stain; has long axis and short axis - Long axis - Aligned with direction of stain - Defines two possible flight paths for the stain - 75-90° angle of impact is elliptical - May have spines and satellite stains - As angle decreases, spines are more prevalent to one side - Width and length of stain is used to estimate angle - Divide width by length and the resulting ratio is equal to the arcsine of the angle of impact - Below 40° degrees - More elliptical and the outflow of the stain produces a single satellite stain - Blood trails - The combination of gravity and momentum cause the droplets to hit their target surface at varying angles - Shapes may give speed and direction - As speed of horizontal movement increases, each droplet falls with greater forward momentum and angles of impact more acute, creating stains more elliptical - Increased horizontal speed also increases horizontal air resistance - Vertical drop distance increases, the forward moving drop encounters air resistance for longer time which counts forward movement of blood, pushing blood to opposite direction, decreasing angle of impact with stains less elliptical - Wipes and swipes - Evidence of motion is more obvious with wipes (also indicates direction) - Swipes are not as obviously and depends on how the object met target - Direction of motion may be defined by assessing the thinning of the stain's appearance or color - Feathering - Thinning accompanied by trailers of stain leading away from the parent stain left behind as the bloody object loses contacts with the target surfaces - Often seen with hair swipes - Feathering on one side shows direction - Smear - Large volume of blood that is distorted so that further classification is impossible - Similar to smudge but w/ more blood - Smudge - Bloodstain distorted so that no classification can be given - Swipe - Transfer of blood onto a surface not already containing blood - Wipe - Bloodstain pattern created when an object moves through an existing blood stain - Transfer pattern repletion - Used to determine occurrence of motion at scene - Hands, feet, or socks - Continues until blood sources is depleted - Those left later in the series are usually more distance and easier to determine - Flow patterns - Flow of blood - Irregular flows may be due to capillary action or flow is dammed by object - Pattern transfer - Transfer of blood from one bloody object to another surface resulting in a patter from the first object - Created by footwear, hair, or hands - Often overlooked - Evaluation - Approach 1 - Look for unique pattern - Look for distance patter or defect in stain - Compare this with class characteristics of standards - Look for class matches and try to individualize if possible - Exact matches are unlikely - Compare with range of sources - Get others' opinions - Approach 2 - Create range of standards for comparison (often created with suspected items) - Compare with stains and look for class characteristics - "screen positive" samples are compared by making transparencies of the object and stain then evaluating using light box and overlaying transparencies - Hair swipes - Classic transfer patterns - V-like (bifurcations) - Pools and standing blood - Passive flow - Flow created by gravity alone with no circulatory action involved and influenced by body position - Active flow - Occurs when body is still living and bleeding occurs - Drying time, volume of blood, and clotting time can indicate things about a stain - Drying time - A dried ring forms a wet blood droplet within 50 seconds - An outer ring remains even if the stain is wiped away - Drying time is dependent on nature of target surface and environment of stain - Skeletonized blood stains - When a dried bloodstain begins to flake in the center leaving a visible outer rim or if the central area of partially blood stain is wiped - Clotting time - Indication of bloodshed and discovery time - 3 phases - Initiation - Formation - Retraction - Separation of serum from fibrin clot - Occurs 30 min. to 1.5 hr - Affected by temp, humidity, nature of target surface and air movement - Clot formation begins 5-20 min after blood shed - Volume - Help determines whether a victim could have survived - Factors to help determine amount of blood loss - Surface characteristics, amount of congealed blood, and absorbency of surrounding area - Wet volume estimates - Recreate a similar stain, measures area of stain and then estimates volume within the stain - Clothing and fabrics - Passive deposits - Transfer, flow patterns, saturation stains and drips - Active deposits - Impact spatter, arterial spurts expirated stains and cast off - Analysis - Area covered in white butcher paper - Determine best stains for analysis - Stain clothing from victim should show grouping characters of the victim but errant blood spatter within a larger pattern may be the assailants - Do not submit samples with mixed stains - Stains recovered for serology tests after stain patterns examined and recorded - Splashed blood - Occur when large volumes of blood (\>1 mL) meet low velocity forces or fall freely to a surface - Have large central areas and elongated peripheral spatters - Secondary spatter - Ricochet; occurs when blood is defected from one surface to another - Can occur if victim is bleeding severely - Drip patterns - Result in blood dripping into blood which occurs when a person stays in one position long enough to drip - Two distinct characteristics - Random satellite spatter that surrounds the pool - Formation of an inverted fan shape on an adjacent vertical surface - Satellite spatters - Detached from the bloom and projected outward creating small spatters - Radiate outwards several feet from pool - Wave effect created in pool when drop has fallen - As droplets detach from blood, they follow traditional parabolic flight paths and impact any vertical surface - The base of the fan is the point closest to dripping actions - Provide evidence of continual motion - Projected blood - Blood is released or projected in large volumes when impacting impinging forces of gravity - Ex. Vomited blood, arterial spurting, or patterns when someone runs through pooled blood - Peripheral spines and narrow streaking - Ricochet - Deflection of large volumes of blood after impact w/ target surface - "Secondary splash" - Types of spatters - Spatter is the random distribution of variable sized bloodstains produced by a variety of different mechanism - Impact spatter is a bloodstain pattern created when a blood source is impacted with a blow or force resulting in the random dispersion of smaller drops - Depends on volume of blood of original source and degree of force - proportional to the amount of energy imparted on stain - Categories of spatter sizes - Mist - Produced when blood is a fine spray - Occasionally referred to as atomized blood\\ - From explosive force - Less than 0.1 mm - Fine - Greater than 0.1 but less than 1 mm - Small - Greater than 1 but less than 3 mm - Medium - Greater than 3 but less than 6 mm - Large - Greater than 6 mm in diameter - Categories for methods of description for velocity - Low velocity - Large stains; 4 mm or \>; result from normal gravitational forces; non-spatter stains included in this classification - Causes: passive drops falling to a close surface; nature blood flow; venous injury - Medium velocity - 1-4 mm; result of blood sources impacted with velocity 5-25 ft/sec; beatings and bludgeoning - Causes: beatings and stabbings involving glass, knives, fist, brick, and baseball bat - High velocity - \>1 mm; force w/ velocity greater than 100 ft/sec. mist like; gunshots or high sped machinery; travels short horizontal distance - Causes: gunshots, explosions, high speed vehicular injuries - Pattern Configuration - Impact spatter - Radiating patterns - Elliptical stains - Created when the target lies parallel to the primary direction of force within the impact - Spherical stains - When the target lies perpendicular to force - Air resistance - Inverse effect on droplets based on size - All droplets projected at same speed will have different flight parabolas, with smaller drops encountering more air resistance and not traveling as far as larger droplets - Important in high velocity impacts - Other effects on spatter pattern - The way an item impacts the contact site and shape of weapon - These effect how blood ejects from impact site and the angle of the weapon may prevent spatter to spray symmetrically - Dispersion characteristics - Lack of spatter on a perpetrator due to being behind zone of radiating pattern - Lack of spatter does not prove lack of presence or participation - Provide info that relates to distance the spattered target was from the impact and verify proximity of an object - Gunshot impact spatter - High velocity spatter - Mist like spots less than 0.1 mm in dia. - Size range - Depends on distance to target, amount of blood, the weapon caliber, the location, number of shots and presence of clothing or hair - Might be like high velocity impacts like explosions, machinery and high-speed vehicular injuries - Droplets radiate outwards in a three-dimensional pattern (cone) - Causes of spatter w/ gunshot - Attributed to formation of wound cavity caused by physical crushing of tissue as the bullet passes - Causes a temp cavity to form in the wound making tissues stretch and collapse - The collapse of the cavity occurs after projectile enters and exits - For multiple shots, the first wound bleeds in-between shots - The second shot makes the first wound compress and squirt out contents - The closer the wounds, the more dramatic the effect - - Sources - Entrance wound - Blowback or back spatter - Found on weapon and the assailant's hands or arms - Less defined due to primary force of the impact bacing projected in the direction of the projectile - Absence of blowback depends on distance and manner in which weapon was held - Exit wound - Forward spatter - As the bullet or fragments exit the body, blood and tissue are carried with it - More symmetrical than back spatter - The closer the target the more spatter produced - Close targets lack dispersion due to size of the mist of droplets and may look spray painted - Further targets produce fewer mist like stains - Beating and stabbing spatter - Medium impact spatter - The first impact rarely produces a spatter - Once wound is open and sufficient blood to produce pooling, spatters will occur with consecutive blows - Number of blows and weapon type affect spatter - Produces spatter with 1-3 mm diameter - Produces satellite and secondary spatter patters from blood dripping onto surfaces - Multiple spatters can occur - Combination of beating, gunshot, arterial spurting, expirated blood and secondary and satellite staining and range of pattern seen may overlap - Secondary and satellite spatters - A single drop of blood hitting a rough surface will produce small satellite stains called secondary spatter - Blood dripping into blood on a horizontal surface will produce drip patterns - Patterns tend to be large and irregularly shaped - Surrounded by small circular or oval satellite spatters around the periphery created by small spatters separating form the main bloodstain at the moment of impact - Splash pattern is a stain w/ a dia. of 0.1 mm or more, created by a low velocity impact against a surface - Spines: pointed edge characteristics that radiate away from the center of a bloodstain and formation depends on impact velocity and surface texture - Spatter and contact stains - Viewing stains - Using a low mag stereoscope, and analyst can see blood traces on the upper surface of the weave of the fabric - Deeper areas of fabric weave do not have any blood staining due to swiping - Spatter will permeate into the weave - Back spatter droplets are found in weave - Cast off stain - As a weapon is swung back and forth, centrifugal forces cast residual blood on the weapon onto any close by surfaces - Due to centrifugal forces as the blood object is swung in an arc and from inertia at the end of the swing - Linear stains that are larger than impact blood spatters - Can be used to estimate number of blows and if the instrument was swung with the left or right hand - Seen with hammers and baseball bats - Assault weapons - Different weapons have distinct effect on results patterns - A broad object will create a pattern different than a sharp or single edge - Sharp weapons - Linear small spatters - Broad weapons - Broad with disperse stains - Blood volume - Amount of blood on source affects cast off pattern - Orientations - Cast-offs give info relating to orientation or nature of blow and whether blow was applied from left or right direction - After initial blow, the second hit will cause a cast off to hit a surface at a 90° angle - After that the following blows with have angles of less than 90° - Positions of victims - Stains may begin on high surface targets then move to low targets indicating the victim was standing then changed positions - Cast off can also come from victim moving arms and legs around - Expirated blood - Blood that is forcefully expelled from the mouth or nose of a living victim due to blood accumulating in lungs or airways from trauma - Creates fine spatter to those resulting from medium or high velocity impacts - Can be heavy, large stains or mist like stains - May include air bubbles and diluted with saliva or nasal mucous - Mimics gunshot spatter - Less vividly colored - Fly spots - Result from fly activity - When flies feed on blood - As they move, they trace and regurgitate blood and leave small marks that are pattern like - Very symmetrical when regurgitated - Specks test positive for blood with presumptive tests - Arterial spurting - Due to blood exiting the body under pressure from a breached artery or the heart - Can help determine physical status of victim - If stains are lacking, or weaker spurts are observed, the victim may be dead or have a decreased heart rate once the artery was hit - Depends on size of damaged artery - Stain can range from small fine spray to gushing or spurting - Amount of blood - Depends on severity of damage, size of vessel, position of victim, and presence of clothing - May appear repetitive - Due to heart beating as blood is expelled under pressure - Altered stains - Clots of blood on clothing or at scene may show drag patterns - Indicates the movement of body or infliction of further injury after initial attack - Can be from coughing or exhalation of clotted blood - Wet stains altered - From smearing, smudging, or wiping - Can also be due to medical personnel treating victim - Dilution - By rain, snow or cover-up attempts - Heat and fire - Mask bloodstains or destroy them - Voids - Patterns that lack staining - "ghosting" - Shows up when another target surface overlays or intervenes with primary target surface - May suggest nature of object or shape of item - Identified by the presence of abrupt and defined edges to the pattern - Useful for establishing body position of victims or assailants - Bubble ring - When blood containing air bubbles dries and retains bubble shape - Weapons - Blood transfer to different weapons - Knife - Can have a lack of blood due to the blade being "cleaned" as it pulls back through tissue layers - Assault weapon - Recording blood spatter - Notes - Form color - Direction of splash - Estimated height of fall - Photos - Best mechanism to record and preserve appearance of blood - Should show overall stain and close-up views should include