SPDX311: SPECIAL DIAGNOSTIC PROCEDURES

TOPIC: FORENSIC SCIENCE

2nd SEMESTER | S.Y 2024-2025
LECTURER: Prof. Rose Dyane Nunag-Hizola, RMT, MPH
TOPIC experience and coursework in law and criminal
SUBTOPIC procedure.
SUB SUBTOPIC • Bachelor's degree programs also included
opportunities for hands-on training in forensic
science laboratories.
INTRODUCTION TO FORENSIC SCIENCE • In recent decades, graduate degrees, particularly at
the master's level, have become more common.
• Forensic science describes the science of associating • Graduate programs typically require a bachelor's
people, places, and things involved in criminal activities; degree in a science field and focus on applying
these scientific disciplines assist in investigating and scientific principles to forensic work, along with
adjudicating criminal and civil cases. coursework in law, criminal investigation, and
- A detective work that uses science; criminal justice.
- For example: someone gives their DNA in an event • Research is also a significant component of graduate
that there’s a crime, we can detect their fingerprints programs.
and we can identify who is/are the possible • For more information about forensic science
suspects for that event, etc. education accreditation standards, refer to the "In
More Detail: FEPAC" resource.
FORENSIC SCIENTIST’S ROLE
• Forensic scientists have two major duties:
- Performing scientific analysis of evidence
- Offering expert testimony in criminal and civil
proceedings
• There are sometimes other responsibilities, such as:
1. offering training in evidence collection and
preservation
2. research
3. validation of procedures for advancing new
methods

THE FORENSIC SCIENTIST (EDUCATIONAL

REQUIREMENTS)
• Historically, forensic scientists were often recruited FORENSIC SEROLOGY OVERVIEW
from chemistry or biology majors without specific • Forensic serology is a crucial part of forensic science,
education in forensic sciences. used for examining crime scenes and analyzing evidence
• Since the mid-20th century, colleges and universities in laboratories.
in the United States have started offering
• It helps identify bodily fluids such as blood, semen,
undergraduate and graduate programs in forensic
saliva, and urine, laying the groundwork for further
science.
• Undergraduate programs initially provided a strong forensic analysis.
foundation in chemical, mathematical, biological, and • Main approach:
physical sciences, along with practical laboratory → Focuses on identifying stains
→ Ensures samples are preserved for future testing

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 EQUIPMENTS USED IN FORENSIC SEROLOGY
ESSENTIAL FORENSIC SEROLOGY FIELD KIT
COMPONENTS
• Personal Safety & Documentation
• Personal Protective Equipment (PPE): Coveralls,
disposable nitrile gloves
• High-resolution camera, scale bar, and identification
labels (for photographs)
• Crime scene tape
• Location recording device
• Site recording form
• Unique sample identifiers
• Logbook and pen
• Permanent marker pens
COLLECTION AND MEASUREMENT TOOLS
• Stainless steel spade and/or trowel
• Spatulas (varying sizes or disposable plastic)
• Measuring tape or yardstick
SAMPLE STORAGE AND DECONTAMINATION
• Sample storage containers (tube, sacks, vials, etc.) THE MAJOR BODY FLUIDS
• Evidence bags
• Deionized water • Blood
• Sterilizing liquid (e.g., Virkon® or alternatives for • Semen
cleaning tools between samples) • Saliva
• Paper towels
BLOOD
• Sample collection vials or pots with Teflon caps or
aluminum cap inserts • In forensic serology, the ABO Blood Group is the genetic
marker commonly used in the identification of blood.
CONSIDERATIONS FOR SPECIMEN COLLECTION AND
TRANSPORT

PRESUMPTIVE TESTS FOR BLOOD DETECTION

• These tests react with hemoglobin present in blood.
• Purpose: To indicate the possible presence of blood ata
scene.

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 THREE MAIN PRESUMPTIVE TESTS: SEMEN
• Colorimetry PRESUMPTIVE TESTS FOR SEMEN DETECTION
• Fluorescence
• Based on the detection of acid phosphatase (an enzyme
• Chemiluminescence
found in high concentration in semen).
COLORIMETRIC TESTS FOR BLOOD DETECTION • Most common test: Brentamine Fast Blue B, applied to
a sample on an alpha naphthyl phosphate substrate.
• The testing chemical is applied to the suspected stain,
• Positive Reaction: Purple color
followed by an oxidant, usually 3% hydrogen peroxide.
• These are called catalytic color tests because CONFIRMATORY TESTS FOR SEMEN
hemoglobin catalyzes the reaction, causing a color
• Also involves acid phosphatase detection, but confirms
change.
the presence of sperm cells.
• Traditional method: Christmas Tree Stain
• Positive Reaction:
- Sperm head → Pink
- Midpiece → Blue
- Tail → Yellow-green
- Skin cells → Green to blue-green

REAGENTS FOR LARGE-SCALE SEROLOGY TESTING

LUMINOL
• Very sensitive to hemoglobin – detects blood at
dilutions of 1 in 5,000,000.
• Does not interfere with DNA analysis (PCR).
• Produces chemiluminescence (glows in the dark).
FLUORESCEIN
• Prepared similarly to luminol but contains a thickener
in commercial versions.
• Adheres better to surfaces, ideal for walls or vertical
areas.
• Produces fluorescence when illuminated at 450 nm
(blue light).

CONFIRMATORY TEST FOR BLOOD

• Based on the formation of crystals using heat and
chemicals.
• Takayama Test (also known as Hemochromogen Test)
- A sample of the presumptive stain is placed under a
cover slip.
- The sample is then briefly heated and viewed under
a microscope SALIVA
- Positive result: Formation of salmon-colored
crystals confirms the presence of blood. • Importance in Forensics
• Found in:
- Bite marks

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 - Licked adhesives (envelopes, stamps) • This is done at a crime scene, to reconstruct the process
- Food and drink surfaces of events that occurred
- Areas with spitting (expectoration)
• Detection challenges:
TERMINOLOGIES
- Often invisible or faint • Passive Bloodstain - patterns created from the force of
gravity (clots, drops, flows, and pooling.)
TESTING FOR SALIVA
• Salivary amylase is the main target
- Present in saliva but not exclusive (also in other
fluids)
- Positive = Presumptive, not confirmatory
• Former test: Radial diffusion (rarely used today)
• Current protocol:
- Positive amylase tests → DNA analysis
• DNA Typing • Transfer bloodstains: wipes, swipes, pattern transfers,
- Saliva contains many epithelial cells and general contact bloodstains
- Enables reliable DNA profiling - Wipe Stain - created when an object moves through
a pre-existing bloodstain (e.g., a clean rag moved
through a pool of blood).
- Swipe Stain - transfer of blood onto a target by a
moving object that is itself bloodstained (e.g., a
BLOODSTAIN PATTERN ANALYSIS bloodied rag being moved across an unstained
floor).

• Projected Bloodstains or Impact Bloodstains - are

described as patterns resulting from blood that is
projected through the air by an external force, such as
blunt force, gunshot, or stabbing.
- Spatter - a technical term describing stains resulting
• Bloodstain Pattern Analysis (BPA) involves: from blood hitting a target.
- Analysis and interpretation of: - Forward Spatter - blood droplets projected away
o Dispersion from an item.
o Shape characteristics - Back Spatter - blood droplets being projected
o Volume toward the item.
o Pattern - Cast-off Stain - blood being flung or projected from
o Number a bloody object in motion or one that stops
o Relationship between the stains suddenly.

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 POINT OF ORIGIN
• "Whenever the direction of a bloodstain can be
determined, it can be expected to have originated at a
point somewhere along that line."
• Can demonstrate a convergence of lines (paths),
indicating the point of origin.
• Presumptive serological tests can be employed to
discover if the stain in question is truly blood.
• By finding the path for each bloodstain in a pattern, the
analyst can interpret a point-of-origin. The more paths
that converge, the greater the likelihood that this is the
• Arterial Spurts and Gushes - occur when an artery is actual origin.
breached while the heart is pumping; produces a zigzag, • However, analysts must be cautious of multiple nearby
up-and-down pattern. impact events. Just because points appear to converge
• Voids - an indicator that some secondary object came doesn’t always mean that this is the true point-of-
between a blood spatter and the final target; leaves an origin—context and pattern differentiation are critical.
outline or "shadow" on the final target.

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 DOCUMENTATION FORENSIC DNA ANALYSIS
1. Document the entire scene as discovered, including DNA [DEOXYRIBONUCLEIC ACID]
'establishing' photographs.
• Deoxyribonucleic acid (DNA) is a molecule found in
2. Photograph pattern transfers, pools, and other fragile
nearly all cells.
patterns first.
3. Document patterns with 'establishing' photographs that • It is a polymer, meaning it is composed of repeating
show the pattern’s relationship to landmarks or other simpler units called monomers.
items of evidence. • DNA is located in two regions within a cell:
4. Take macro and close-up photographs; include a scale in - Nucleus - Nuclear DNA/Genomic DNA
every photograph. - Mitochondria - Mitochondrial DNA
5. When reconstructing the point-of-origin, document
individual stains used in the reconstruction.

STRINGING
• Easiest and cheapest method to interpret bloodstain
patterns
• A string is run from the presumptive source to the
bloodstain
• Push pins or masking tape are used to hold string ends
in place
• Tools like rulers, protractors, or laser pointers help align
the string
• Purpose:
- Determines the point-of-origin NUCLEAR DNA
- Visualizes the angles of impact
- Shows where multiple stains converge • Nuclear DNA is found in a geometric shape called a
• How it works: double helix.
- Attach strings to the narrow ends (tails) of stains • It is made up of alternating sugar molecules
- Pull them back into 3D space (deoxyribose) and phosphates.
- The intersection point is the possible source of • Attached to each sugar molecule is one of four bases
blood (nucleotides):
• Limitations of Stringing: - Adenine (A)
- Best on flat surfaces - Guanine (G)
- Needs clear, defined stains - Cytosine (C)
- May be inaccurate in complex or 3D scenes - Thymine (T)

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 DNA TYPING DNA AMPLIFICATION
RESTRICTION FRAGMENT LENGTH POLYMORPHISM • Where it’s done:
- Performed using thermal cycler/thermocycler
1. DNA is extracted from biological material.
• Process:
2. It is then cut into small fragments using restriction
- Follows the three main steps of Polymerase Chain
enzymes.
Reaction (PCR):
3. These enzymes target minisatellites—specific repeating
o Denaturation – DNA strands are separated
DNA sequences.
by heating.
4. The length variations (polymorphisms) in these
o Annealing – Short primers bind to the target
minisatellites help differentiate individuals.
DNA sequence.
• In forensic analysis, 4 to 6 highly polymorphic loci are
o Extension – DNA polymerase extends the
typically analyzed for comparison.
primers to form new DNA strands.
DNA TYPING
DNA EXTRACTION
• Process:
1. Lyse cells to release the DNA molecules.
2. Separate DNA from other cellular materials.
3. Isolate the DNA into a format compatible with
downstream applications, including PCR
amplification.
• DNA Storage Guidelines:
- Short-term storage:
o Refrigerator at 4°C
o Freezer at -20°C
- Long-term storage:
o Ultra-low freezer at -80°C
• Important note:
- DNA extraction must be conducted in a physically
isolated location—separate from where PCR • Polymerase Chain Reaction (PCR)
amplification or other downstream applications are - PCR is a technique used to amplify specific DNA
performed, to prevent contamination. sequences.
- Sensitive and prone to contamination, so strict lab
DNA QUANTIFICATION
protocols must be followed.
• Purpose: - DNA extractions are always performed in a
- To determine the appropriate amount of DNA physically isolated location from where the
template to include in PCR amplification of short amplification will occur.
tandem repeat (STR) loci. - Amplification is carried out in thermal cyclers
• Why it matters: (thermocyclers), which cycle through temperature
- Ensures that the data is accurate and within range, changes to complete:
avoiding: o Denaturation
o Off-scale data o Annealing
o Associated artifacts that may affect o Extension
interpretation

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 STEP 1: DENATURATION - Separates DNA fragments according to size —
• DNA is added to the PCR reaction mixture. smaller fragments move faster and travel farther
• The mixture is heated to 95°C, causing the double- than larger ones.
stranded DNA to denature.
• Hydrogen bonds between base pairs break, resulting
in single-stranded DNA.
• Each strand now serves as a template for the
formation of new double-stranded DNA.

DNA PROFILE COMPARISON

• Helps calculate the likelihood of a coincidental match
between DNA from evidence and that of a suspect.
ANNEALING
• Understanding a "Match"
• A short strand of synthetic DNA called primers is
- DNA typing examines only specific parts of the DNA.
attached to each of the separated DNA strands.
• Primers serve as the starting point for adding new - Further testing could reveal differences at untested
bases to reproduce each strand. genetic markers.
• The thermal cycler temperature drops to 60 °C to - Scientists use the term "genetic concordance" to
allow primers to bind to the DNA templates. describe consistency at tested points, while
acknowledging possible differences elsewhere.
• Importance of Communication
- Clear and accurate explanation of DNA analysis
results is essential in legal contexts to prevent
misinterpretation or confusion in court.
EXTENSION
• Reaction temperature is raised to 72 °C.
• Under the action of Taq polymerase, nucleotides
(single bases) are added to the primers.
• This builds the entire complementary strand,
resulting in double-stranded DNA.
• The cycle repeats: the temperature is raised again to
94 °C to start a new round of denaturation.

MITOCHONDRIAL DNA
• Differences between mtDNA and Genomic DNA:
- mtDNA is circular, while genomic DNA is linear.
- mtDNA is more abundant in cells, with many copies,
while genomic DNA has only two copies per cell.
SEPARATION OR IDENTIFICATION - All mtDNA (from both males and females) comes
• Gel Electrophoresis from the mother.
- A technique where DNA fragments are pulled - mtDNA shows a lot of variation between unrelated
people, making it more useful for forensic
through a gel matrix using an electric current.
identification.
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