FSCT 8150 Week 2 - Sample Collection, Storage, and Characterization Notes
Sample Recovery
Forensic ident specialists (Canada) / Criminalists (US) collect evidence at crime scenes, ensuring meticulous documentation and preservation of the scene's integrity.
Police officers collect reference/comparison samples from:
Individuals of interest (suspects, victims) to exclude their DNA through comparison analysis.
Deceased individuals to establish identification and link them to potential crimes.
Other persons of interest to gather a comprehensive range of samples for thorough investigation.
Questioned samples (collected from the scene) are often referred to as "Q samples," representing unknown origin evidence.
Known samples (reference/comparison samples) are sometimes referred to as "reference samples," providing a baseline for comparison.
Packaging of Question Samples:
Small items (clothing, weapons) are placed in evidence bags, sealed and labeled to maintain chain of custody.
Larger items (mattresses, chairs) can be:
Sectioned and a portion submitted, ensuring representative sampling of the item.
Swabbed with water or another solution to collect trace evidence from the surface.
Scraped (e.g., blood from pavement) to gather concentrated samples from hard surfaces.
Known samples are collected using:
FTA cards for direct DNA collection and preservation.
Buccal swabs to obtain DNA from cheek cells.
Liquid blood samples when a larger quantity of DNA is required or for specific testing methodologies.
FTA Cards
FTA cards are used for collecting and preserving DNA samples, providing a stable matrix for long-term storage.
Indicating FTA cards change color (pink to white) upon saliva application for easy visualization, ensuring proper sample deposition.
Samples are typically taken from FTA cards using a punch, creating a small disc containing the DNA sample.
Newer technologies allow direct PCR amplification from FTA card punches, bypassing quantification and extraction steps, saving time and resources.
FTA cards contain chemicals to:
Lyse cells, breaking open cell membranes to release DNA.
Denature proteins, inactivating enzymes that could degrade DNA.
Protect nucleic acids (DNA) from nucleases, oxidation, and UV damage, preserving sample integrity.
Inactivate organisms, including bloodborne pathogens, ensuring safe handling of the samples.
Prevent bacterial and microorganism growth, maintaining the purity of the DNA sample.
DNA can be preserved on FTA cards for extended periods (validation studies show successful recovery after 17+ years at room temperature), demonstrating long-term stability.
Packaging Evidence
Proper packaging by forensic IDENT members is crucial to avoid contamination and degradation, ensuring the integrity of the evidence.
Guidelines:
Avoid plastic packaging when possible; use brown paper bags to allow breathability and prevent moisture buildup.
If plastic is necessary, ensure the evidence is completely dry before packaging to prevent mold and bacterial growth.
Use separate containers for each piece of evidence to prevent cross-contamination, maintaining the integrity of individual samples.
Maintain separation between different scenes and individuals to avoid mixing evidence from different sources.
Brown paper bags are preferred over plastic because they allow the evidence to dry, preventing bacterial growth, condensation, and DNA degradation, preserving the quality of the DNA.
Biological Samples for DNA Recovery
Forensic biologists prioritize sample types with a high probability of DNA recovery:
Blood (DNA from white blood cells), providing a rich source of nuclear DNA.
Semen, containing a high concentration of sperm cells with distinct genetic material.
Saliva, containing epithelial cells and white blood cells with recoverable DNA.
Contact/Touch DNA (sloughed skin cells), often found on handled objects and surfaces.
Hairs with roots, as the root contains nucleated cells with DNA.
Less often: urine and feces, as these contain lower concentrations of DNA and are more susceptible to degradation.
Red blood cells do not contain a nucleus and therefore do not contain DNA, making white blood cells the primary source of DNA in blood samples.
Presumptive vs. Confirmatory Tests
Two types of tests are used to identify body fluids:
Presumptive tests indicate a substance may be present but are not specific, serving as initial screening tools.
Confirmatory tests definitively establish the presence of a substance through specific and reliable methods.
Example: Hemostix is a presumptive test for blood but yields false positives with animal blood, certain metals (copper), suede, leather, grass stains, vegetable matter with peroxidase activity, fecal material, sweat, and saliva, requiring further confirmatory testing.
Presumptive Tests
Presumptive tests are rapid and assist in localizing a body fluid, allowing for quick identification of potential evidence.
They are not specific, requiring additional confirmatory testing to ensure accuracy.
Using presumptive tests (e.g., UV light, Hemostix) can save time by focusing confirmatory tests on specific areas of large items (e.g., king-size duvet), streamlining the testing process.
Alternate Light Sources:
High-intensity light sources (portable) enhance the detection of body fluids and other evidence.
Single or multiple wavelengths can be used to target specific substances.
Used in the lab or at the crime scene for versatile application.
UV lamps emit light in the 10-400 nm range, causing certain substances to fluoresce or absorb light.
OmniPrint alternate light source uses a liquid light guide for precise and controlled illumination.
Blood absorbs UV light (appears darker), while semen, saliva, and urine fluoresce (appear brighter), aiding in their detection.
Goggles are used to visualize fluorescence and protect eyes from UV light; different colored lenses are more effective at different wavelengths, optimizing visualization.
Fluorescence is the emission of light upon absorption of light, with the emitted light having a slightly longer wavelength, a phenomenon used in forensic detection.
Body Fluid Fluorescence:
Semen: High fluorescence, making it easily detectable under UV light.
Blood: Absorbs light, appearing dark under UV illumination.
Urine: Weaker fluorescence than semen, requiring careful observation.
Saliva: Similar fluorescence to urine, making differentiation challenging.
Hair: Usually does not fluoresce, providing a contrast against other fluorescent materials.
Fibers: Can absorb or fluoresce depending on the fiber, requiring careful examination.
Biochemical Tests for Blood
Presumptive Tests:
Color-Producing Tests: Chemical reaction with blood results in a color change, indicating the presence of blood.
Light-Producing Tests: Light reaction indicates a positive result (e.g., Luminol, Bluestar), enhancing visualization.
Catalytic Tests: Based on the peroxidase activity of the heme group in blood, utilizing its catalytic properties.
General Peroxidase Activity Reaction:
Heme is the iron-containing compound in blood and acts as a catalyst, accelerating the reaction.
Specific Color Tests
Benzidine Test:
Rarely used due to being highly carcinogenic and less sensitive than other tests, posing health risks.
o-Tolidine Test:
Sensitive but carcinogenic, limiting its use due to safety concerns.
Phenolphthalein Test (Kastle-Meyer Test):
Used by the Center of Forensic Science as a reliable presumptive test.
Sensitive but not specific to human blood, yielding false positives with non-human substances.
False positives occur with oxidizing agents (e.g., peroxidases in cauliflower, broccoli), requiring careful interpretation.
Produces a pink colored reaction, indicating the possible presence of blood.
Tetramethylbenzidine Test (Hemostix):
Very sensitive and less carcinogenic, making it a safer alternative.
Originally designed for urine analysis but adapted for blood detection.
Can be applied directly or indirectly via wet filter paper for versatile use.
Positive result: green color change, indicating potential blood presence.
Luminol
Used to screen large areas for blood, especially at crime scenes, enhancing detection capabilities.
Viewed and photographed in the dark to capture the luminescence.
Extremely sensitive; reacts even with cleaned-up bloodstains, detecting trace amounts.
Not specific for human blood; reacts with bleach, requiring careful interpretation.
May dilute or wash away trace evidence, potentially compromising other evidence.
Limitations: False positives can occur with hypochlorite, microbes, or vegetable matter, necessitating cautious analysis.
Confirmatory Tests for Blood
Crystal Tests (Takayama Test):
Based on preparation of hemoglobin derivatives with specific chemicals, confirming blood presence through crystal formation.
Viewed under a microscope for detailed analysis.
More labor-intensive and not always as sensitive as presumptive tests; many labs no longer perform these due to efficiency concerns.
Hematrace Test:
Immunological test similar to a pregnancy test, specifically identifying human blood.
Determines if blood is of human origin, differentiating it from animal blood.
Sample is lysed, and liquid is applied to the test strip for analysis.
Two lines indicate a positive result for human blood (test and control lines), confirming its presence.
One line (control line only) indicates a negative reaction, ruling out human blood.
Used to differentiate between human and animal blood, aiding in species determination.
Takayama Test:
Produces pink crystals, confirming the presence of hemoglobin.
Developed in 1912, a classic confirmatory test.
More specific but less sensitive than peroxidase tests, requiring careful execution.
Can be challenging with old blood stains, potentially affecting accuracy.
Tests for Semen
Semen is a mixture of cells, amino acids, sugars, salts, ions, and other organic/inorganic materials, providing a complex composition.
Ejaculate volume is typically 2-6 mL, with hundreds of millions of sperm per milliliter, varying among individuals.
Tests for Sperm:
Fast Blue B Test: Presumptive color test for acid phosphatase from the prostate, indicating potential semen presence.
PSA/P30 Test: Presumptive immunological test for prostate-specific antigen (PSA), a marker for semen.
Crystal Formation Tests: Rarely performed (Florence and Barberio tests) due to complexity and availability of alternative methods.
Visualization Test: Confirmatory test using microscopy (Christmas tree stain or newer staining methods), directly observing sperm cells.
Acid Phosphatase Test
Produces an insoluble purple color in the presence of semen, indicating the presence of acid phosphatase.
Acid phosphatase concentration is 100 times greater in semen than other tissues, making it a strong indicator.
Potential for layering of body fluids leading to false positives, requiring careful sample preparation.
False positives: vegetable matter, vaginal secretions, fecal matter, fungus, contraceptive foams, and acid phosphatase in nature (but at much lower levels), necessitating cautious interpretation.
Fast and simple test for screening large areas, quickly identifying potential semen stains.
PSA/P30 Test
Presumptive test for semen, a commercial product by ABA company, widely used in forensic labs.
Very sensitive, detecting even small amounts of semen.
False positives: vaginal secretions, breast milk, amniotic fluid, requiring careful consideration of context.
False negatives: High-dose hook effect (excessive semen concentration):
Dilute sample 10x or 100x and rerun on a new card to avoid the hook effect.
Previously considered confirmatory but now regarded as presumptive due to false positives, requiring further confirmation.
Mechanism:
P30 (prostate-specific antigen) protein in semen binds to mobile anti-human P30 antibody with pink dye in the sample well.
Complex moves up the card due to wicking action.
If semen is present, the complex binds to immobilized anti-human P30 antibodies at the test line, creating a pink line.
Excess antibodies continue to the control line and bind to immobilized anti-immunoglobulin antibodies, creating a second pink line.
Both lines must be present for a positive result, ensuring proper test function.
In the high-dose hook effect, only the test line appears (failed test requiring dilution), indicating the need for dilution and retesting.
Confirmatory Test for Semen (Microscopy)
Visual identification of sperm cells using a microscope, directly confirming semen presence.
Sperm has a head containing nuclear DNA and a flattened oval body, providing distinct morphological features.
Identifiable with contrast staining and the acrosome (top of the head), enhancing visualization.
Sperm Structure:
Head (nucleus with nuclear DNA), containing the genetic material.
Midpiece (mitochondria with mitochondrial DNA), providing energy for sperm motility.
Tail and end piece, facilitating sperm movement.
During fertilization, only the sperm head enters the egg; the tail falls off, transferring genetic material.
Mitochondrial DNA is inherited from the mother (egg) and not the father (sperm), providing lineage information.
Christmas Tree Staining:
Sperm heads stain pink/red/purple, enhancing their visibility.
Tails stain yellow-green, differentiating them from other structures.
Midpiece stains blue, highlighting this region.
Challenges: epithelial cells can obscure sperm visualization, requiring careful observation.
Sperm Highlighter:
Stains sperm green and epithelial cells blue for clearer identification improving contrast.
Specific for human sperm, ensuring accurate identification.
Allows for better sperm counting for DNA extraction success, optimizing downstream analysis.
Laser Microdissection:
Sperm highlighter used in combination with laser microdissection microscopes, enabling precise collection.
Allows for precise cutting and collection of sperm cells (e.g., 25-50 sperm), minimizing contamination.
Avoids female DNA contamination for more effective DNA extraction, improving DNA profiling.
Semen Screening Flowchart
Different levels of tests (positive or negative) lead to different conclusions about sperm presence, guiding the investigation.
Semen may be present without sperm if the individual has undergone a vasectomy:
Microscopy will be negative, but PSA/P30 tests will be positive, requiring careful interpretation.
Multiple tests are useful in these cases, ensuring accurate assessment.
Saliva
Saliva is an alkaline secretion containing water, mucus, protein, salts, and enzymes, aiding in digestion.
Humans produce 0.5-1.5 liters per day, essential for oral health and digestion.
Used for digestion, breaking down food particles.
No specific confirmatory test for saliva; instead, tests for alpha-amylase, an enzyme present in high concentrations.
Amylase concentration is typically 50 times higher in saliva than other tissues (presumptive test), making it a reliable indicator.
Saliva Tests:
Amylase Test (Starch-Iodine Test).
Phadebas Reaction.
Starch-Iodine Test
Iodine-starch appears blue; amylase breaks down starch, diminishing the blue color, indicating amylase presence.
Test diffusion on agar gel plate with wells, allowing for controlled reaction.
Suspected saliva samples are loaded into wells, enabling amylase diffusion.
Iodine is added after incubation to show starch digestion progress, visualizing amylase activity.
Amylase presence visualized as a circular void against the blue background, indicating starch breakdown.
Phadebas Reaction
Phadebas is starch bound to a dye molecule; the starch complex is insoluble, facilitating amylase detection.
Amylase in saliva separates the starch from the dye, releasing the dye.
Two tests: tube test and press test, providing versatile application.
Press Test:
Filter paper coated with insoluble starch complex is pressed against the evidence item, capturing amylase.
In the presence of amylase, the free dye is released and diffuses through the filter paper, indicating amylase presence.
Visualized as blue color on the other side, providing a clear indication.
Mapping: The distance from the edge indicates the location of saliva/amylase for cutting samples for DNA extraction, guiding sample collection.
Phadebas is highly selective for alpha-amylase, minimizing false positives.
Sweat, semen, and vaginal excretions do not give positive results within 10 minutes, whereas saliva will, ensuring specificity.
Fecal stains also have high levels of alpha-amylase, requiring careful interpretation.
Summary
Common questions in forensic biology: Is this blood? Is this semen? Is this saliva?, guiding the investigation.
Negative test: No further DNA testing may be warranted, saving resources.
Positive test: Sample may continue for DNA testing, progressing the investigation.
Overlap (rare exceptions): Negative test, but DNA testing may still be necessary if there's a concern about touch DNA or if the male has a vasectomy, ensuring thoroughness.
Why Do Biological Body Fluid Testing?
Forensic DNA testing is human-specific, focusing on human DNA profiles.
The answer depends on lab-specific factors: validated methods, caseload, types of cases, influencing testing decisions.
In some cases, it may not make sense to test for saliva (e.g., straw and pop can), saving time and resources.
In other cases, it may be very important to establish that the DNA came from semen (e.g., sexual assault case), having significant legal implications.
Common Questions
Is this blood? (motor vehicle accident investigation: airbag, window visor), aiding in accident reconstruction.
Human salivary amylase is present in urine, blood, semen, and breast milk, but the level in saliva is significantly higher (40 times stronger than in breast milk), allowing for differentiation.
Is saliva present? It can be one of the more difficult answers because there's no confirmatory test for saliva; at best, we can infer from the strength of the reaction, requiring careful analysis.
Is this