Forensic Characterization of Bloodstains using Digital Imaging Bloodstains

Forensic Characterization of Bloodstains using Digital Imaging • Bloodstains that are on an item that’s dark or has a complex pattern can be difficult to see • A digital imaging system with an infrared filter attached can make bloodstains more visible by filtering out the background color

Forensic Characterization of Bloodstains cont’d • Micro Crystal Tests – Takayama – Tiechman • Advantages – More specific then chemical test • Disadvantages – Not as sensitive – More susceptible to interference

Forensic Characterization of Bloodstains: Individualizing • ABO Blood Typing • Enzyme Typing • DNA Analysis Ancient – RFLP: Restriction Fragment Length Polymorphism – STR: Short Tandem Repeats

Forensic Characterization of Semen: 1. VISUALIZATION MANY BODY FLUIDS FLUORESCE WITH ALTERNATE LIGHTING SOURCES

Forensic Characterization of Semen: 2. PRESUMPTIVE TESTING ACID PHOSPHATASE ENZYME / FOUND IN LARGE CONCENTRATIONS IN SEMEN

Forensic Characterization of Semen: 3. CONFIRMATION A. MICROSCOPIC IDENTIFICATION B. DETECTION OF P 30, A MALE OF SPERM PROSTATE PROTEIN. USEFULL FOR VASECTOMIZED MALES

Intact spermatozoa on a vaginal slide

Forensic Characterization of Saliva • Evidence commonly tested for the presence of saliva includes: Detection of Amylase (breaks down starch in gel) – Cigarette butts – Envelope flaps – Swabs taken from the body of sexual assault victims – Bottles, cans, & straws Gel contains starch that is broken down (circles ) in the presence of Amylase enzyme

Safety First—safeguards while handling biological evidence • Wear gloves • Keep contaminated surface away from face —protect those mucous membranes • Properly dispose of gloves/wash hands

Goals of biological evidence collection • Collect as much sample as possible from a single source—keep it concentrated • Ensure that the sample is not inadvertently mixed with other biological samples—change gloves if contaminated • Handle the sample in a manner that minimizes deterioration—air-dry quickly (and no heat or sunlight exposure)

Further recommendations for collection of biological evidence • Handle as little as possible—submit the item with the stain still on it • If stain is on a large porous surface (e. g. , a rug) cut out the stain area (plus unstained) • Collect with slightly moistened (with dist. water) cotton swab—keep it concentrated, and take a control swab too

Taking care to avoid contamination • Don’t allow one evidence stain to come into contact with other biological samples, including transfer from tools and gloves • Don’t talk or cough over evidence stains • Collect and package stains separately • Clean tools (e. g. , tweezers) thoroughly, with distilled water stream, dry with tissue, repeat • May use disposable tools

Packaging biological evidence • Allow stains to air-dry as much as possible before placing in paper bag or envelope—do not use plastic • Use separate paper containers for each item and package stains and controls separately • Ensure that the paper container is large enough to allow circulation around the evidence item • For garments, use clean paper to prevent different stains from contacting each other

Proper collection and procedures are critical to avoid Contamination How else will you detect it and reduce/avoid contamination?

Typically, it looks like a mixture: A combination of DNA from two/more persons

Contamination at autopsy—teeth from decomposed body

Contamination at autopsy— mixed reference

Contamination at autopsy— resolution

Contamination in the lab From sample with high level of DNA To sample with low level of DNA

PCR Product Contamination— the Thousand to One Nightmare It only takes a minuscule amount of amplified product… …to cause a typing disaster

Contamination—Prevention • Sample items one-at-a-time • Separate evidence samples from reference samples • Use protective gear • Separate work areas with dedicated equipment

Monitoring for Contamination— Controls ‘R’ Us Bloodstain (Evidence) Substrate Control Reagent Blank—for Evidence Victim’s Reference Sample Reagent Blank—for References Negative Amplification Control Quality Control Sample Positive Amplification Control

Summary 1 • Screening samples for biological evidence includes both physical (e. g. alternate light sources) and chemical (presumptive tests) detection methods. • 3 steps- Visualization, Presumptive testing and Confirmation – Blood detection include observation of color, microcrystalline tests, luminol detection, chemical presumptive tests based on hemoglobin's peroxidase-like activity, antibody species tests, and DNA analysis. – Semen detection includes observation of fluorescence, Acid phosphatase detection methods, then microscopic examination, P 30 testing and DNA – Saliva detection includes microscopic examination, amylase detection and DNA

Summary 2 • Safety- In collecting biological evidence, safety is paramount. Wear gloves- away from face- Properly dispose of gloves/wash hands • Collect as much sample as possible - keep it concentratednot inadvertently mixed -change gloves if contaminated • Handle the sample in a manner that minimizes deterioration—air-dry quickly (and no heat or sunlight exposure) • Prevent contamination- Sample items one-at-a-time. Separate evidence samples from reference samples- Use protective gear- Separate work areas with dedicated equipment- Clean the area, tools and run QC • Monitor for contamination with controls

I. Intro to DNA : Facts and Jargon DNA: Deoxyribonucleic acid Different in every *individual The same in every **cell of an individual's body *except for identical twins that have the same DNA - "The time honored method of cloning humans" ** diseased individuals may be mosaics

DNA Facts and Jargon Review Where is it? How is it stored? DNA is found in every *cell= basic unit of life Inside nuclei (organization center for the cell) and mitochondria (ATP powerhouse of the cell) & chloroplasts for plants- (making our food via photosynthesis) Nuclei are not found in red blood cells In white blood cells, saliva, skin, hair fingernails, urine, feces, vomitus, earwax etc.

DNA in the Cell In nuclei, mitochondria and chloroplasts (plants) organized in chromosomes (wound around histones) “DNA double bagging” DNA Spaghetti Metaphor chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides

DNA function What’s it do? Deoxyribo. Nucleic Acid : blueprints of life Replication, Information Storage and Mutation- RIM Central Dogma information flow--------> DNA------->RNA------>protein transcription translation Like Phone numbers- Units the same- Difference is in the Sequence

http: //www. ncbi. nlm. nih. gov/genome/guide/ DNA Organization and Inheritance Human Genome Contains 23 Pairs of Chromosomes It is inherited from your mom and dad 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Sex-chromosomes

Where’s Daddy? PCR product size (bp) 11 12 8 14 14 11 12 8 14 12

DNA Structure What is it? Bases (AGCT) form the stairs of the ladder, are faithfully paired and exhibit differences. P S-A : T-S P P S-G : C-S P P S-A : T-S P P S-G : C-S P Hydrogen bonds 5’ 3’ 5’ A=T G C hybridized strands 3’ T=A Phosphate-sugar backbone T=A C G A=T T A T G C G C G G C 3’ A=T T=A A denatured strands C 5’ 3’ G C Sugars (S) and phosphates (P) form the sides of the ladder (identical for all DNA). Bases (AGCT- Asian Guys Can Teach) form the stairs of the ladder, are faithfully paired by hydrogen bonds and exhibit differences. A : T and G : C (DNA is where its AT)

DNA Structure • Primary genetic material is composed of two complementary strands • Form a double helix or twisted ladder • Sides are sugar phosphate and the steps are base pairs • Four Bases- 2 Purines – Adenine and Guanine and 2 Pyrimidines- Cytosine and Thymine • Asian Guys are Pure!

DNA Structure Nucleotides are the building blocks themselves composed of PBS Nucleotides-PBS the only channel I want my kids to watch Phosphate (negative charge) Base (AGCT-Asian Guys Can Teach) Sugar (deoxyribose-5 C) Phosphate-Sugars Connected by phosphodiester linkages

DNA Structure 2 Complimentary, Antiparallel Strands held together by Base Pairs- H Bonds A: T held with 2 H Bonds . G: C held with 3 H Bonds

Molecule of Heredity Limerick Steve Lee The molecular structure today Is heredity’s DNA With nucleotides completely comprised of a sugar and phosphate and base Adenine and thymine can base pair Forming two hydrogen bonds for one stair Cytosine and guanine pair with three in between and are equal in size when compared The bases you see are so keen They include thymine and adenine Cytosine and one more with guanine can store all the info with rungs in between DNA strands are just not the same One is coding and one is called lame (anticoding) They are opposite in direction and this is called antiparallel in name The sides of the ladder you know, are sugar and phosphate which show that Franklin was right double helix is tight ten base pairs per turn in a row Complimentary nature of strands lets replication proceed just as planned with A paring to T and G pairing to C the fidelity is precise and quite grand

• DNA Structure/Function Teaching Metaphors and Acronyms Cell Biology – Spaghetti in Bags • Function: – RIM- Pacific Rim – Information Storage- Phone Number analogy • Structure: – PBS- The only station I want my kids to watch – Asian Guys Can Teach: AGCT, Asian Guys are Pure – DNA is where its AT – Hybridization- Battleship, DNA velcro (David Letterman) – Repeat Sequences- Trains- number of box cars