LEARNING OBJECTIVES

• Understand the difference between presumptive and confirmatory tests for body fluid identification.

• Be aware of the limitations of presumptive tests.

• Be able to describe the basis for and compare the techniques used in the identification of blood, semen, and saliva.

• Have an understanding of the theoretical basis of using RNA as a target for identifying the body fluid of origin.

INTERACTIONS BETWEEN THE THREE COMPONENTS OF THE CRIMINAL JUSTICE SYSTEM

• Diagram based on John M. Butler (2009) illustrates the interactions between:
    - Law Enforcement
      - Police Agencies (local, state, federal)
      - Laws and police training
      - Investigators/Detectives
      - Evidence submitted

• Scientific Analysis
      - Forensic Laboratory
      - Validated scientific tests
      - CSI
      - Other Forensic Disciplines
      - DNA Unit
      - DNA Analysts
      - Scientific report(s) completed

• Legal Proceedings
      - Court System
      - Legal framework and precedent
      - Judge, Prosecution, Defense
      - Conviction or trial/exoneration
      - Evidence returned to law enforcement after trial

• Research introduces new methods.

FORENSIC BIOLOGICAL SPECIMENS

• Common biological specimens collected in forensic investigations include:
    - Semen
    - Blood
    - Skin
    - Saliva
    - Bones
    - Hair
    - Less common exhibits: Urine, Feces, Vaginal Secretions, Vomit.

TYPES OF DNA SAMPLES COLLECTED

• Unknown samples from crime scenes.

• Elimination samples from individuals (victim(s) or family members) who had prior lawful access to the crime scene.

• Samples from Police, CSI Officers, and DNA lab personnel (elimination database).

• Biological material abandoned by known individuals.

SAMPLE COLLECTION

Collection of Reference DNA Samples

• Collected from:
    - Suspects or convicted felons and victims.
    - Family reference samples for cases such as paternity testing, missing persons, and mass disaster victim identification.
    - Buccal cell collection involves cotton swab inside the cheek.
    - For children, a disposable toothbrush can be used.
    - Samples must be DRY or collected using a treated collection card.

SWABS

• Various swabs available for DNA collection:
    - Cellulose, cotton.
    - Nylon flocked swab.
    - Treated cards (FTA).
    - High-Performance DNA collection systems such as:
      - Isoheli Swabs
      - High Yield Swabs
      - DNA Isolation & Stabilization Kits
      - DNA Purification Kits

• Drawbacks of cotton fibers:
     - Tend to entrap cheek cells, leading to lower yields in DNA analysis, increasing chances of retest.
    - Advantages of nylon flocked swabs:
      - Cheek cells adhere better to the nylon fiber and elute more readily due to capillary action.

DOUBLE SWAB TECHNIQUE

• Double Swab Technique involves:
    1. Wet swab to rehydrate cells.
    2. Dry swab to collect cells.

• Clothing/Fabric collection: Use adhesive tape attached to plastic or acetate support ('tape-lift').
    - Options include Dissolvable tape and Free Zond.

SAFEGUARDING DNA EVIDENCE AND YOURSELF

• Biological material can contain hazardous pathogens, such as the hepatitis A virus.

• To protect the integrity of evidence and health of personnel:
    - Always wear gloves and change them frequently.
    - Use disposable instruments or clean instruments thoroughly.
    - Avoid touching areas where DNA might exist.
    - Avoid talking, sneezing, or coughing near evidence.
    - Avoid touching face during evidence collection.
    - Air-dry evidence thoroughly before packaging.
    - Place evidence in new paper bags or envelopes; do not use plastic bags or staples.

CONTAMINATION CONCERNS

• Cross-contamination from tools:
    - Brushes used to dust for fingerprints can cross-contaminate samples.
    - A study showed that 51 used latent fingerprint brushes provided a full or partial DNA profile 86% of the time.

• Transfer Types:
    - Primary and secondary transfer rates were documented:
      - Porous substances/dry samples (0.36%)
      - Moist with absorbent materials (2%)
      - Plastic (95%).

• Example of contamination is the Phantom of Heilbronn, a German unresolved investigation from 1993 to 2009.

REAGENTS AND PLASTICS

• ISO 18385:2016 ensures products used for collecting, storing, and analyzing biological material for forensic purposes are designed to minimize the risk of human DNA contamination.

PRESUMPTIVE VS. CONFIRMATORY TESTS

Presumptive Tests

• Use of Alternate Light Sources (ALS) for initial detection and screening of potential biological evidence.

• General Workflow includes:
    - Evidence Photograph
    - ALS application (stains darken or fluoresce)
    - Tests like Phenolphthalein and Acid Phosphatase indicate positive or negative results.

• Blood Detection:
    - Reacts with blood but not specific to blood alone.
    - Not 100% conclusive; relies on the catalytic properties of blood hemoglobin with peroxidase-like activity.

• Colorimetric Tests for blood include:
    - Tetramethylbenzidine (TMB) Test
    - Kastle-Meyer Test.

Kastle Meyer Test (Phenolphthalein)

• Reagent reactions to the iron in hemoglobin using hydrogen peroxide:
    - Formula: $ext{Fe}^{4+} + ext{Phenolphthalin} ext{ (colorless)} + ext{H}_2 ext{O}_2 <br>ightarrow ext{Phenolphthalein (pink)} + ext{H}_2 ext{O} + ext{Fe}^{3+}$

• Color visible as bright pink upon oxidation of phenolphthalin.

Chemiluminescence and Fluorescence Tests

• Both types produce light, used when expecting blood presence but are not visible:
    - Examples include Luminol and BlueStar tests; they emit a blue glow.

• Caution: Can interfere with DNA analysis; do not use if stains can be collected directly.

BLOOD CONFIRMATORY TESTS

• Tests that are specific to human blood identification include the Precipitin Test and Ouchterlony double immunodiffusion.

• Current tests:
    - ABA Hematrace test kit: high sensitivity and specificity for human blood using monoclonal antibody.
    - RSID Blood Kit detects glycophorin A instead of hemoglobin.
    - ABA HemaTrace detects human hemoglobin >0.05 µg/ml but has known false positives with primates.

PRESUMPTIVE TEST FOR SEMEN

• Detection of acid phosphatase (AP): high concentration in human semen, basis for testing.

• Reaction with sodium alphanapthylphosphate produces a purple-blue color.

• AP Test involves:
    - Moisten filter paper/cotton swab
    - Apply to questioned stain
    - Add reagent
    - Development of purple color indicates semen.

SEMEN CONFIRMATORY TEST

• Identifies sperm cells using microscopic techniques:
    - Nuclear Fast Red (Christmas tree stain) for staining:
      - Human sperm head stained crimson, tail stained green-blue-gray.

• Considerations for absence of sperm (aspermia or oligospermia).

• Confirmation through detection of P30, a prostate-specific antigen only found in significant quantities in human semen.

• ABAcard p30 test is the method of choice with potential for false positives from other body fluids.

SALIVA TESTING

• Saliva aids initial digestion and contains alpha-amylase:
    - Presumptive test for amylase is not specific to saliva.

• Phadebas™ Reagent Test determines salivary amylase by color reaction:
    - Color production proportional to amylase concentration.

LIMITATIONS OF PRESUMPTIVE TEST FOR SALIVA

• Amylase found in other body fluids (e.g., feces).

• Repeated depositions can give misleading amylase levels.

CONFIRMATORY TEST FOR SALIVA

• Lateral flow strip tests specific for human salivary alpha-amylase antigen without cross-reactivity with other fluids.

FUTURE OF BODY FLUID IDENTIFICATION

• Significant body fluids and tissues of forensic relevance include:
    - Blood, Menstrual blood, Saliva, Semen, Skin, Vaginal secretions.

• Importance of differentiating between tissue types in forensic and legal contexts.

ALTERNATIVE BODY FLUID IDENTIFICATION TECHNIQUES

• Focus on nucleic acid tests (e.g., mRNA, miRNA expression).

• Examining patterns of gene expression unique to cell types for body fluid identification.

DNA METHYLATION BODY FLUID IDENTIFICATION

• Methylation modifies gene expression patterns; provides tissue specificity.

• CpG islands show tissue-specific methylation patterns useful for forensic differentiation.

ADDITIONAL READING

• Recommended chapters for further study: Chapters 5 – 9 from Forensic Biology (2015) by R. Li, CRC Press. https://doi.org/10.1201/b18209

The notes cover key aspects of forensic biology including:

Learning Objectives

• Understanding the difference between presumptive and confirmatory tests for body fluid identification, their limitations, and techniques for identifying blood, semen, and saliva using RNA as a target.

Interactions in Criminal Justice

• Explores the interactions between law enforcement, scientific analysis, and the legal proceedings involved in forensic investigations.

Biological Specimens

• Common specimens collected include semen, blood, skin, saliva, bones, and hair, along with methods for DNA sample collection, cross-contamination concerns, and sample integrity.

Detection and Testing

• Presumptive tests utilizing alternate light sources, colorimetric methods for blood, tests for semen (acid phosphatase), and saliva (alpha-amylase) are covered, along with more specific confirmatory tests for blood, semen, and saliva.

Future Techniques

• Discusses advancements in body fluid identification techniques, including nucleic acid tests and DNA methylation as emerging methods of forensic differentiation.