Forensic Techniques: Entomology, Soil, DNA, Single‑Cell Analysis

Ian Macaulay, a molecular and cellular biologist at the Earlham Institute in Norwich, introduced an evening dedicated to crime-solving using advanced genomic technologies. He explained that a few years prior, scientists like himself collaborated with professional forensic scientists to explore new applications of genomics in understanding crime scenes, DNA transfer, and persistence, aiming to enhance the justice system.

The scenario for the evening involved an unknown deceased female found in a woodland, with her identity, cause, and time of death unknown. The presentation aimed to demonstrate how various forensic techniques could be applied to such a case.

Forensic Entomology: Estimating Time Since Death

Dr. Cat Brown, a forensic entomologist from the University of Portsmouth, was introduced to discuss how insects can help determine the time of death. She highlighted that insects are particularly useful for estimating time since death more than 72 hours after the event, when other pathological indicators like rigor mortis, liver mortis, and algor mortis have subsided.

Upon examining the simulated crime scene, Dr. Brown noted the presence of large larvae, indicating that the death occurred beyond the initial 72-hour window. She observed larvae in typical locations such as underneath the cadaver and in natural orifices, which are moist and humid, ideal for egg-laying. She also found larvae around the neck, which she described as unusual and potentially indicative of neck injury or bruising, a detail that would be important for a pathologist.

Insect Collection and Analysis

Dr. Brown demonstrated the collection process using a Petri dish and forceps, emphasizing the need to collect representative samples from all different areas without disturbing other evidence. Half of the collected larvae would be reared to adulthood, and the other half preserved for microscopic examination.

For preservation, larvae are quickly killed using boiling water, then sieved and placed in 70% ethanol to prevent degradation and shrinking, allowing for accurate measurement of their length and characteristics. Adult insects are also collected to identify species.

Estimating Time of Death from Insects

Entomologists prefer to be called to the scene to understand the environmental context. Insects detect the smell of a cadaver within minutes and lay eggs within a few hours. By examining the larvae under a microscope, specific features can be identified. Dr. Brown pointed out posterior spiracles, which maggots use for breathing. The number of slits (three in this case) indicates the larval stage (third stage). The size, shape, and distance of these spiracles, along with the structure of the oral sclerite (mouth hooks used for grip, not biting) and the pattern of spines on the body, help identify the species.

In this case, the initial findings suggested the common species Calliphora vicina (bluebottle fly). These flies have a life cycle similar to butterflies: adults visit the body, lay eggs, which hatch into maggots, then pupate, and finally emerge as adults. The observed third instar larvae were nearly finished feeding and starting to wander away from the body.

By correlating the length of the larvae with the temperature at the scene, the age of the larvae can be estimated. At approximately 20°C, these larvae were estimated to be about 6 days old.

Additional Information for Confidence

Dr. Brown stressed that this initial estimate is crude. To refine it, environmental conditions such as temperature, wind speed, rainfall, sunlight, cloud cover, humidity, and the specific environment (woodland vs. field) are crucial. These factors influence how quickly insects arrive and develop. For instance, if it's been very wet, flies might not fly, affecting the initial colonization time. Considering all factors, the minimum time since death was estimated to be around 6 days.

Beyond entomology, other forensic evidence would be collected, such as apparent blood on the head, unusual seeds and leaves not native to the area, and soil. This led to the introduction of a forensic soil scientist.

Forensic Soil Analysis: Tracing Locations

Professor Lorna Dawson, a soil expert, explained how soil can be a source of evidence. She noted that the soil on the victim's feet appeared different from the sandy soil at the discovery location (Fred Wells Gardens, near the Thames in central London).

Soil Collection and Initial Assessment

The first step is to identify potential points of contact for control samples, especially if a suspect is apprehended. The SOCO (Scene of Crime Officer) team would collect samples from these locations. The soil on the victim's feet was finer-textured than the coarse sandy soil at the scene.

Using sterilized equipment, the soil is photographed, recovered from the footwear, and placed in an evidence bag, treated like any other evidence.

Laboratory Analysis of Soil

Soil analysis involves understanding its physical, chemical, and biological characteristics. A CT scan can reveal the soil's structure, showing distinct pads. Comparing the questioned sample with known soil types (clay, sand, silt) helps classify it. In this case, the questioned soil resembled silty loam.

Particle size distribution, determined by shaking soil in water and observing settling rates (Stokes' Law), further confirmed the silty nature. This allowed the exclusion of clay and sandy soils, narrowing down potential locations.

Chemical Analysis and Elemental Signatures

The chemical composition of soil, including elements like aluminum, silica, cobalt, cadmium, iron, and copper, provides a unique fingerprint. By analyzing the concentration of these elements, specific areas can be identified. For example, mapping zinc concentrations and overlaying them with aluminum, chromium, and cobalt data can pinpoint a precise location. This process of overlaying elemental maps helps exclude areas that don't match, leading to a specific red-squared area.

Artifacts and Vegetation: Further Pinpointing

Within the soil, artifacts like fluorescent fibers can be found. In this case, tiny fluorescent fibers were discovered, matching the database profile of road worker vests. This suggested recent roadworks in the area. Combining this with the 6-day time window from entomology, a specific roadwork site north of Berkeley Square, where work had occurred about a week prior, became a strong candidate.

Additionally, vegetation found in the victim's hair, specifically a distinctive hairy seed pod from a plane tree, provided further clues. Overlaying this information with the identified location revealed a row of plane trees on the north edge of Berkeley Square, strongly suggesting this as a secondary or even primary site where an incident might have taken place.

Forensic Genetics: DNA Evidence

Dr. Nick Dorne, a forensic geneticist from Liverpool John Moores University, discussed the collection and analysis of DNA evidence. At the secondary scene (north end of Berkeley Square), various types of evidence would be collected:

• Marks and Traces: Footprints, fingerprints from cans or handled items.
• Biological Evidence: Red staining on a baseball cap (potentially blood), and a rope (ligature) found with the cap. The rope could contain cells from the handler and the victim.
• Other Items: A discarded screwdriver.

Prioritizing Evidence Collection

The CSI team prioritizes evidence that is transitory (likely to change over time). Footwear marks, for example, are collected first as they can degrade quickly due to weather. These marks can be linked to the make and model of footwear.

Collecting Footwear Marks

Footwear marks are collected using Denstone, a plaster of Paris-like material mixed with water and poured into the impression. This creates a hard cast that preserves all features, including sole patterns and wear and tear, which can then be compared to footwear databases. Each piece of evidence is secured in specialized packaging, taped, and labeled with an evidence tag detailing the exhibit reference number, description, time, date, location, and crime reference number. A chain of custody log ensures that every handler of the evidence is recorded.

DNA from Evidence

DNA can be obtained from various items:

• Touch DNA: From handled items like the screwdriver, due to the shedding of skin cells.
• Rich Biological Sources: Blood on the cap, saliva on the rim of a drinking vessel.
• Other Characteristics: Potential tissue on a tissue, or bloodstains.

Contamination Control and Packaging

To prevent contamination, CSI teams wear scene suits, face masks, and PPE, which also protects them from potential biohazards. Different items require specific packaging, such as weapons tubes for sharp objects like screwdrivers, to prevent damage to the packaging and preserve the evidence.

Success Rates of DNA Profiling

Dr. Dorne presented statistics on the success rates of generating usable DNA profiles:

• Blood: Approximately 89% of the time, yielding high-quality profiles with distinct, high-peak alleles.
• Saliva: About 63% of the time, with slightly lower peak heights due to less DNA.
• Touch DNA (e.g., screwdriver): Only 12% of the time, often resulting in poor quality profiles with missing information and reduced peak heights.
• Cord (ligature): A mere 8% of the time, highlighting the significant challenges with touch DNA on such items.

Understanding Touch DNA

Touch DNA, also known as trace DNA, is the genetic material left behind when someone touches an object. Research suggests that the amount of DNA transferred is largely independent of how long an item is held; the initial contact transfers a significant amount. However, this amount is orders of magnitude less than what is found in blood or saliva.

Forensic Biochemistry: Analyzing Stains and DNA

George Zouganelis from the University of Derby discussed the biochemical analysis of stains and DNA profiling. He emphasized the importance of verifying the nature of a stain before proceeding with expensive DNA tests.

Presumptive Blood Test (Kastle-Meyer Test)

Using the red-stained baseball cap, Zouganelis demonstrated the Kastle-Meyer test, a presumptive test for blood. A small amount of the stain is transferred to filter paper, wetted, and then sprayed with KM solution, ethanol (as a negative control), and hydrogen peroxide. If blood is present, it immediately turns purple. This meticulous, step-by-step approach is crucial to avoid accusations of evidence planting.

DNA Analysis Findings

After verifying the presence of blood and other biological materials, DNA analysis is performed:

• Blood on the hat: Matched the victim's DNA, indicating she was wearing the hat during the incident.
• DNA from the Coke can: Matched an alleged suspect already in custody.
• DNA from the screwdriver: A complex mixture containing the suspect's DNA plus two unknown individuals.
• DNA from the ligature: Hair matched the victim's, and tissue samples showed a similar mixture to the screwdriver, implicating suspect D.

Challenges with DNA Mixtures

Zouganelis highlighted that these successful outcomes represent the 10% of cases where things work perfectly. In reality, DNA mixtures are a significant challenge. If results are not clear, it can lead to scenarios where:

• The suspect's DNA is present, but the two unknowns are different people.
• The mixture contains DNA from three irrelevant people.

These complexities make interpretation difficult, and communicating such data in court is challenging due to the intricate mathematics involved. This led to the idea of analyzing individual cells.

Advanced Technologies: Single Cell Analysis for DNA Intelligence (SCANDI)

Ian Macaulay returned to discuss how molecular biology advancements could address the challenge of DNA mixtures. He explained that DNA profiling relies on analyzing specific regions of the human genome (STRs) to create a unique "barcode" for each individual. The UK's National DNA Database, the world's first, contains about 7 million such profiles.

The Problem of DNA Mixtures

Crime scene samples often contain DNA mixtures from multiple individuals due to the sensitivity of current methods and the constant shedding of cells. Besides the suspect and victim, DNA from other individuals (e.g., someone who handled the ligature at a store, or someone who shook hands with the suspect) can accumulate.

Macaulay demonstrated this with a mixture of colored sweets representing cells from different individuals. A conventional DNA extraction from this mixture results in a "horrible brown mess," making it impossible to identify individual contributors. Mixtures account for 45-66% of DNA profiles in European national DNA databases.

SCANDI Project: Single Cell Analysis

The SCANDI (Single Cell Analysis for DNA Intelligence) project, funded by UKRI, aims to overcome this by focusing on individual cells. Inspired by biomedical advances in understanding the 37 trillion cells in the human body, the project views cells as the vehicle for DNA transfer.

Humans constantly shed billions of cells from various sources:

• Shedding: Mouth, gut epithelial, dandruff, hair.
• Touch: Epidermal cells left every time something is touched.
• Intimate Contact: Sperm cells, epithelial cells.
• Injury: Small or large numbers of cells transferred.

The principle is that "every contact leaves a trace," and cells are the vehicle for this trace. If an individual cell contains a nucleus, it holds all the genetic information needed to identify a person. By analyzing individual cells from a mixture, distinct profiles can be obtained.

Cell Sorting and DNA Profiling

The project uses fluorescent-activated cell sorting (FACS) to pick individual cells from a mixture. This machine can sort thousands of cells in parallel, taking pictures of each cell to identify its type (e.g., sperm cells vs. epithelial cells). From these single sorted cells, a full, database-searchable DNA profile can be generated.

Macaulay presented anonymized data showing that this method can generate high-accuracy DNA profiles from single cells, effectively "unmixing" complex samples. This technology is transitioning from theoretical possibility to practical application.

Impact on the Justice System

The ability to resolve DNA mixtures has significant implications for the justice system:

• Preventing False Convictions: By providing clearer, unambiguous DNA evidence.
• Maximizing Evidential Value: Utilizing samples that might otherwise be uninterpretable.
• Stakeholder Importance: Everyone is a stakeholder in the justice system, and investment in such technology is crucial.

Macaulay emphasized that while much academic research focuses on "interesting" topics, this project addresses a truly "important" issue. He noted that violent crime affects more people than many major diseases, highlighting the societal value of investing in forensic technology. All the techniques demonstrated, from entomology to single-cell analysis, originated from academic research and have been translated into forensic practice.