May 4, 2020 by Locard's Lab

Smoker or Non-Smoker? Donor Characteristics from Saliva

The discovery of body fluids during forensic investigation can provide countless clues to a crime. Saliva is often countered during certain investigations, particularly in the investigation of sexual assaults and other violent crimes. The wealth of chemical information housed in the complex matrix could provide clues as to who might have left the substance behind. One example of this is whether or not the donor is a smoker.

A team of researchers at the University at Albany have hit the news multiple times in recent years with their rapid, on-site tools for body fluid analysis. Using Raman spectroscopy, the group have successfully developed methods to identify body fluids, estimate body fluid age, and determine characteristics of the donor, such as race and sex. In the latest paper by Igor Lednev and his colleagues, published in Journal of Biophotonics, Raman spectroscopy has been shown to differentiate between smokers and non-smokers.

Working in collaboration with researchers at Kuwait University, the team applied Raman spectroscopy to dried saliva from smokers and non-smokers, aiming to use chemical differences in the samples to determine whether or not the donor smokes. Raman spectroscopy is a non-destructive technique that enables the rapid, on-site analysis of samples, producing distinctive chemical fingerprints consisting of bands produced by the interaction of light with molecular structures.

One might assume the test would target nicotine, a major chemical component in tobacco. However, nicotine is relatively short-lived in the body, thus is not a suitable target for analytical tests. Instead, the researchers focused on cotinine, a primary metabolite of nicotine with a notably longer half-life. Saliva samples from 32 donors were analysed by Raman spectroscopy and the chemical profiles produced studied for differences. Researchers soon encountered a problem. Raman bands indicative of cotinine overlapped with typical Raman bands produced by saliva, making the detection of cotinine in saliva challenging. The team used machine learning to solve this problem.

First, they identified eight spectral regions that contributed the most variation between the saliva of smokers and non-smokers. Using an artificial neural network, a classification model was constructed for the prediction of smoking habits of a donor. By inputting chemical data from known samples, the network is able to learn from the data in order to predict an output (in this case, whether or not the donor of a saliva sample was a smoker). In laboratory-based studies, the model constructed achieved an impressive accuracy of 100%.

Although this pilot study was based on a very limited sample size, the technique shows great promise in the determination of donor characteristics from dried body fluids.

Al-Hetlani et al. Differentiating smokers and nonsmokers based on Raman spectroscopy of oral fluid and advanced statistics for forensic applications. Journal of Biophotonics, 2019, DOI: 10.1002/jbio.201960123 https://onlinelibrary.wiley.com/doi/abs/10.1002/jbio.201960123

May 17, 2019 by Locard's Lab

Interview with Lecturer in Forensic Science Dr Kayleigh Sheppard

Dr Kayleigh Sheppard works as a lecturer in forensic science at Liverpool John Moores University.

What is your current job role and what does this entail?

I currently work as a lecturer in Forensic Science at Liverpool John Moores University. My role is divided between lecturing undergraduate and postgraduate students, supervising undergraduate and MSc research projects and conducting research. I teach students across a range of undergraduate courses including BSc Forensic Science, BSc Forensic Anthropology and BSc Policing with Forensics, as well as postgraduate students on the MSc Forensic Bioscience course. Across these courses, I deliver a range of lectures and practical sessions focusing on topics such as Crime Scene Investigation and Forensic Methods with a particular focus on the photography of crime scenes and the evidence contained within them. Photography techniques covered include crime scene photography using natural light and flash, and more advanced photographic methods such as oblique lighting, alternate light source photography and automated 360◦ photography. I introduce the topics and theoretical principles of each topic to the students through lectures and workshops and then give the students hands on experience and the opportunity to develop their practical skills for each of the techniques through practical classes.

The practical classes delivered consist of fingermark enhancement, recovery and comparison, footwear mark recovery, evidence packaging techniques and crime scene documentation and photography. In addition, the students put together everything they have learnt throughout the semester and demonstrate their crime scene investigation techniques using simulated crime scenes that we are able to mock up within our crime scene houses. I supervise a range of student projects at both undergraduate and masters level which investigate advanced photographic methods of crime scenes, using 360-degree photography or mobile technology.

What initially attracted you to your particular field of research?

I have always had an interest and passion for the sciences, particularly biology and chemistry and knew that my future career would be in a scientific field. Whilst at school I was a keen problem solver and enjoyed reading crime and true crime novels. The combination of these traits led me to investigate a potential career in forensic science and so I started my BSc in Forensic Science at Staffordshire University. Throughout the course I was particularly interested in the crime scene aspects and envisaged myself going on to work as a crime scene investigator in the future. Upon completion of my course I had the opportunity to undertake a placement with Staffordshire Police. The placement allowed me to put my knowledge from my degree into practice, alongside crime scene investigators, whilst also providing me with the opportunity to conduct a research project. This project focused on my interest in crime scene investigations whilst incorporating emerging technologies- another interest of mine. The project was entitled “Next generation crime scene recording and forensic data use within criminal investigations”. The project was so well received by the Forensic staff that I wanted to pursue this area further and applied for a PhD investigating the use of 360-degree panoramic photography in a forensic context at Staffordshire University.

Alongside my PhD I was able to teach undergraduate students, introduce them to 360◦ camera technology, and provide them with hands on experience using the technology. The ability to apply my research into the curriculum to enhance the students learning sparked my interest in academia. An academic position provides the best of both worlds, allowing me to pass on my knowledge and experience to the students and teach them about forensic science, whilst also allowing me to continue to pursue my own research avenues. It is very rewarding to teach the students about modern and emerging technologies to assist with criminal investigations and to see their enthusiasm about a topic they may not have been introduced to before. The best part about being a lecturer is having the ability to teach students about topics they are unfamiliar with and pass on that knowledge. The most gratifying part of my job is when a student does not understand a topic or does not enjoy it, but through explanation and discussion using different learning techniques, the students understand the topic and begin to enjoy it.

Can you tell us about the research you’re currently involved in?

Most of the research that I conduct investigates the use of 360◦ panoramic photography for documenting and presenting crime scenes. At first, the research sought to validate the technique, regarding its accuracy for taking measurements at a scene. The research has begun to adapt the technology to answer specific research questions, which may aid crime scene investigators at the crime scene, by adapting the technology to make it do something that it could not do before. For example, the 360◦ camera has been adapted to include alternate light sources for the detection of biological fluids, which are invisible to the naked eye, to simultaneously detect and document them in situ at a crime scene. Further research has also looked into the extent to which modern technologies for documenting crime scenes have been utilised for the presentation of evidence in the courtrooms and the factors that may be affecting the use in courtrooms.

The use of alternate light sources has also branched into other research avenues within the forensic field. Current research being conducted investigates the importance of cleanliness and prevention of cross contamination within Sexual Assault Referral Centres (SARC). The issues with identifying contamination in SARC environments is that in order to ensure cleanliness, the contaminants would ideally be visible. Many biological fluids are invisible to the naked eye and therefore we cannot see them – so how do we know whether they are present on a surface or not? Most biological fluids fluoresce under specific wavelengths of light and enable them to be visualised. Research currently being conducted is seeking to determine the effectiveness of a SARCS-LED light source (CopperTree Forensics Ltd.) for identifying human blood, semen, saliva and vaginal secretions in small volumes (less than 1 μl) on a variety of surfaces typically encountered in SARC facilities. A SARCS-LED enables staff to ‘see’ biological traces, so provides a more targeted forensic clean. This layered approach alongside current ATP testing, and improved cleaning methods, can help to deliver a more thorough service. Using such a light source to identify biological fluids or contamination should enable a more effective cleaning protocol to be employed within SARC facilities, providing a more robust anti-contamination process which is in line with the Forensic Regulator expectations.

Semen and vaginal secretions deposited onto a white vinyl surface. Left – observed under natural light and the biological fluids are not visible to the naked eye. Right – observed under a blue SARCS-LED (445 nm wavelength) and demonstrating biological fluid fluorescence.

What are some of the biggest challenges in your area of research?

Academia can be a challenging place to work and trying to make sure that you maintain the knowledge of the forensic science field whilst it is continually updating can be challenging and often involves lots of reading to stay current, as well as attending training courses and conferences. High profile criminal court cases in forensic science are particularly interesting as they demonstrate to the students the importance and real world impact of their degree and the work they will be conducting in the field, so it is important to stay on top of these as well. At such an early stage in my academic career, being only 26, I felt as if there was a lot of pressure to prove myself worthy. As a result, I take advantage of every opportunity that is presented to me to further my knowledge and experience. It can be challenging to maintain a balance of lecturing, creating engaging and interesting sessions for the students, whilst continuing to conduct research and publish within the field. What keeps me going is my passion and enthusiasm for the subject area and the fact that I can shape the minds of the future.

Finally, do you have any advice for young scientists eager to pursue a career in your field of work?

For any individuals who want to pursue a career as a forensic scientist and get involved with any area of forensic science, make sure that you know what to expect. If you are simply going into this field because of your love for CSI: Crime Scene Investigation on the television that is not enough. The field of forensic science is not always as glamourous as it is often portrayed in the media, and some of the analysis techniques are not always conducted at the drop of a hat. However, saying that, forensic science is such an interesting and exciting field that is constantly evolving – no two days will ever be the same.

If you are interested in pursuing a career in this area you will need to make yourself stand out from the crowd. Over the past few years, this is a field which has become extremely competitive and you need to be able to demonstrate that you are a more suitable candidate than everyone else – what makes you different, what makes you stand out? In order to do this I would highly recommend getting any work experience that you can within the area. Working within criminal investigations can be tricky with active casework, but you do not know unless you ask. Some universities have partnerships with the local police forces so make sure to take advantage of any opportunities they can offer you. If this is not possible, try to get experience in laboratories to demonstrate your ability to follow protocols, work to standard operating procedures and avoid contamination. Alternatively, you could volunteer as a special constable within the police or assist within other police departments. Many of the skills that you obtain from these experiences can be transferred into the forensic field and more importantly demonstrates your commitment to enhancing your knowledge and skill set.

Website: LJMU Kayleigh Sheppard

Twitter: @Kay_Sheppard1

January 24, 2017 by Locard's Lab

Sex Determination with Raman Spectroscopy

The ability to quickly identify a victim or suspect during a criminal investigation is crucial, and the use of fingerprinting and DNA profiling often proves invaluable in this. However, a fingerprint or DNA profile can only be associated with an individual if there is an alternative profile or database match for comparison.

But what can investigators do when comparison profiles are not available, rendering biological fluids found at crime scenes somewhat useless?

The capability of instantly establishing alternative information relating to a suspect – such as sex, age or a phenotypic characteristic – based on the analysis of the evidence could be a substantial benefit to an investigation.

In recent years, the use of both well-established and novel analytical techniques to ascertain information relating to a suspect or victim from bodily fluids has been the focus of a great deal of research. With an increasing number of analytical instruments becoming field portable, the possibility of in situ analysis at crime scenes and instant suspect information is quickly becoming a reality.

Raman Spectroscopy and Sex Determination

Most recently, researchers at the University of Albany (Muro et al, 2016) have highlighted the possibility of using portable Raman Spectroscopy to determine the sex of an individual based only on their saliva in real-time.

The study utilised a total of 48 saliva samples from both male and female donors of multiple ethnicities, depositing the samples onto aluminium foil and drying overnight. Samples were then subjected to Raman analysis and the chemical signatures scrutinised to determine whether or not the saliva of male donors differed from that of female donors.

Raman Spectroscopy is a non-destructive analytical technique used for analyte identification based on molecular vibrations. As a basic explanation, monochromatic light is initially directed towards the sample, some of this light simply passing through the sample and some of it being scattered. A small amount of this scattered light experiences an energy shift due to interactions between the sample and the incident light. These energy shifts are detected and transformed into a visual representation. The resulting Raman spectrum typically plots frequency vs intensity of the energy shifted light. The positions of different bands on this spectrum relate to the molecular vibrations within the sample which, if interpreted correctly, can allow for the identification of analytes.

Raman spectra are somewhat characteristic of the chemical composition of the sample. In the case of the saliva analysed in this study, the features of the spectra were largely caused by amino acids and proteins. When comparing the respective spectra from male and female donors, by eye they appear remarkably similar. However using multivariate data analysis, a statistical technique used to analyse data with multiple variables, the researchers were able to distinguish between the saliva of male donors and that of female donors, reporting the ability to ascertain the sex of the donor with an accuracy of an impressive 94%.

Comparison of male and female saliva Raman spectra (Muro et al, 2016)

Although only a proof-of-concept paper, the research demonstrates the possibility of using portable Raman spectroscopy as a method of elucidating donor information, in this case sex, through the analysis of a bodily fluid. The researchers suggest further work will be conducted to include other bodily fluids and donor characteristics.

At this point, the usefulness of the research is limited. Although instantly establishing the sex of the donor of a bodily fluid can aid investigators in developing a suspect or victim profile more efficiently, the pool of potential donors is still huge. The total of 48 saliva donors used in this study is of course not a sufficient representation of the population, thus a much larger sample set would be required to fully evaluate the technique, including non-laboratory setting experiments. Furthermore, there is a wide range of medical conditions and additional factors that can result in changes in the chemical composition of saliva and thus could influence the effectiveness of this technique. Whether or not certain diseases or external influences can hinder gender determination using this method would need to be investigated.

Previous Research

The idea of utilising analytical chemistry to ascertain donor information is not in itself novel, and other researchers have attempted to achieve the same goal through different means.

In 2015, scientists also based at the University of Albany (Huynh et al, 2015) developed a biocatalytic assay approach to the analysis of amino acids in fingerprints to determine the sex of the donor. The study boasted an accuracy of 99%, with the sex differences believed to be due to the higher concentration of amino acids in fingerprints deposited by females.

Research by Takeda et al in 2009 used Nuclear Magnetic Resonance (NMR) Spectroscopy to determine differences between the urine and saliva samples of different donors based on the detection and comparison of different metabolites. Certain compounds, including acetate, formate, glycine and pyruvate, were found in higher concentrations in male samples, allowing for the differentiation between male and female bodily fluids.

The focus of such research is not limited to sex differentiation, for instance some research has even focused on establishing whether a blood sample belongs to a smoker or non-smoker. Utilising gas chromatography mass spectrometry with a solid phase microextraction pre-concentration step, Mochalski et al (2013) were able to effectively distinguish between the blood and breath of smokers and non-smokers due to the ten-fold increase in levels of benzene and toluene, a conclusion which has been repeated by other researchers.

Looking at just this small handful of studies, it becomes evident that certain analytical techniques have the potential power to ascertain a range of information about the donor of a bodily fluid. However all of these immunoassay and mass spectrometry techniques are typically time-consuming, requiring the transportation of a sample to a laboratory, sometimes extensive sample preparation, followed by a form of analysis that will often destroy the sample. This is evidentially not ideal during a time-sensitive criminal investigation in which sample amount may be limited.

To an extent, the research utilising Raman spectroscopy to determine sex from saliva does alleviate some of these problems. The portability of Raman devices allows for in situ analysis, removing the need for expensive and time-consuming laboratory analysis. As Raman spectroscopy is based on the interaction between the sample analyte and light, it is a non-destructive technique, allowing the sample to be preserved for storage and further analyses is required.

Although these techniques do not hold the power of DNA in almost irrefutably identifying the suspect, they may at least aid investigators in narrowing down their pool of suspects and steering the investigation in the right direction. No doubt further advances in analytical chemistry will allow for more accurate and robust techniques in the future.

References

Huynh, C et al. Forensic identification of gender from fingerprints. Anal. Chem. 87(2015), pp11531-11536.

Mochalski, P et al. Blood and breath levels of selected volatile organic compounds in healthy volunteers. Analyst. 7(2013), pp2134-2145.

Muro, C. L et al. Sex determination based on Raman Spectroscopy of saliva traces for forensic purposes. Anal. Chem. 88(2016), pp12489-12493.

Takeda, I et al. Understanding the human salivary metabolome. NMR Biomed. 22(2009), pp577-584.

October 8, 2015 by Locard's Lab

Determining the Age of a Fingerprint: Is It Possible?

During the scrutinising examination of a crime scene, it is entirely plausible for dozens or more fingerprints and fragments of fingerprints to be recovered. Not at all surprising considering how often we touch endless surfaces in our day-to-day lives. Consider how many people might grasp the handle of a shop door in an average day. If that shop were to become a crime scene, how could one possibly distinguish between prints that had originated on the day of the crime and those deposited weeks or months ago? Is it possible to estimate the age of a fingerprint?

Firstly, a quick review of just what a fingerprint is. We all know fingerprints are a series of unique arches, loops and whorls left behind when we touch a surface. But people may be slightly less sure of what these deposits are actually composed of.

Although the composition of a fingerprint is somewhat complex, 95-99% of the deposit is simply water, which will typically readily evaporate. The remaining 1-5% is an intricate mixture of organic and inorganic compounds ranging from amino acids and fatty acids to trace metals. Chloride, potassium, sodium, calcium, hydrocarbons, sterols – the list goes on. A vast concoction of chemicals emitted through our skin and deposited whenever our fingertips touch a surface.

But what we didn’t know until recently, is that these deposited chemicals gradually move with time, and that this movement can be used to determine how long a fingerprint has been on a particular surface. Researchers from the National Institute of Standards and Technology recently stumbled upon this very fact (Muramoto & Sisco, 2015).

Fingerprint when freshly deposited (left) and after 72 hours (right).
Credit: Muramoto/NIST

Like many discoveries, the research itself was something of an accident. The NIST researchers were initially using analytical techniques to detect trace amounts of illicit substances present in fingerprints. In the process of this investigation, they noticed the movement of chemicals within the fingerprint over time. Fingerprints are made up of ridges and valleys forming unique patterns, the characteristic features that allow investigators to distinguish between prints deposited by different people. These features are imprinted in various chemicals when an individual leaves a print behind. However over time the chemicals composing the fingerprint begin to migrate, moving from the defined ridges of the fingerprint into the valleys, essentially blurring the details of the print.

The researchers focused on particular biomolecules, namely fatty acids such as palmitic acid. By depositing fingerprints on sterile silicon wafers and storing the samples under strictly controlled conditions for a period of time, scientists were able to clearly observe the migration of molecules using a technique known as time-of-flight secondary ion mass spectrometry (TOF-SIMS). After a period of only 1 hour after fingerprint deposition, the friction ridge patterns of the fingerprint were clearly visible with the fatty acid molecules under observation residing along the ridges of the print. However within 24 hours the molecules had diffused into the valleys, blurring the patterns of the fingerprint.

The research thus far has simply been conducted to prove the concept of fingerprint component migration for ageing fingerprints, but further work could investigate time effects on a greater scale and even differences in the migration of different molecules. Although the method is advantageous in that it does not depend on chemical changes in fingerprints, which can be very dependent on individual circumstances, further work would be warranted to establish how environmental differences could affect the rate at which this molecular movement occurs, including temperature and humidity effects as well as those caused by the deposition surface.

As intriguing as this research is, this is not the first time scientists have tried to devise a method of ageing fingerprints using chemistry. In fact, researchers have been attempting to accurately age fingerprints for decades. Research has focussed on the changes in the chemical composition of fingerprints over time. For instance, concentrating on a particular compound, such as cholesterol, and establishing the rate at which the concentration of that compound changes over time (Weyermann et al, 2011). Unfortunately many such studies have found changes in the chemical composition of fingerprints to be too variable and unpredictable, particularly when taking into account the differences between donors and the effects of different conditions. Other studies have attempted to determine the age of a fingerprint based on how well powder adheres to the ridges (Wertheim, 2003), by changes in fluorescence wavelength over time (Duff & Menzel, 1978), and changes in electrostatic charge with time (Watson et al, 2010). A vast array of scenarios have been studied intently.

A method of establishing the age of a deposited fingerprint has been at the forefront of latent print research for a long time, and is likely to continue. Although fascinating advances have been made, scientists are a long way from catching criminals by the age of a fingerprint.

References

Cadd, S. Islam, M. Manson, P. Bleay, S. Fingerprint composition and aging: A literature review. Sci Justice. 2015(55) pp. 219-238.

Duff, J. Menzel, E. Laser assisted thin-layer chromatography and luminescence of fingerprints: an approach to fingerprint age determination. J. Forensic Sci. 1978(23), pp 129-134.

Muramoto, S. Sisco, E. Strategies for Potential Age Dating of Fingerprints through the Diffusion of Sebum Molecules on a Nonporous Surface Analysed Using Time-of-Flight Secondary Ion Mass Spectrometry. Anal Chem. 2015(87) pp. 8035-8038.

National Institute of Standards & Technology. Who, What, When: Determining the Age of Fingerprints. [online] Available: http://www.nist.gov/mml/mmsd/20150824fingerprints.cfm

Watson, P. Prance, R. J. Prance, H. Bearsmore-Rust, S. T. Imaging the time sequence of latent electrostatic fingerprints. Proc. SPIE ‘Optics and photonics for counterterrorism and crime fighting VI, Toulouse, 7838, 783803-1-6, ISBN 9780819483560 (2010).

Wertheim, K. Fingerprint age determination: is there any hope? J. Forensic Identif. 2003(53), pp 42-49.

Weyermann, C. Roux, C. Champod, C. Initial Results on the Composition of Fingerprints and its Evolution as a Function of Time by GC/MS Analysis. J. Forensic Sci. 2011(56), pp 102-108.

June 11, 2015 by Locard's Lab

Diatoms and Death By Drowning

A body is found floating in a lake, the circumstances surrounding the death a complete mystery.

One might assume the cause of death to be drowning, and for this there may be certain pathological indicators. But failing these indicators, how can you be so sure that the victim drowned? It could be that they were killed elsewhere, their body tossed into the lake to eliminate suspicion. Or perhaps they did drown, but in alternative circumstances in another body of water. The scenarios are endless. But how can these questions be answered?

The key to this problem might just be a diverse group of microscopic algal organisms known as diatoms.

Perhaps you’ve heard of them. These asexually-reproducing organisms exist in a vast variety of shapes, sizes and colours, plentiful in many aquatic environments and existing in a tremendous range of populations. A particularly important feature of diatoms is their silica-based cell wall, producing an especially distinctive appearance that can vary greatly between different species. This cell wall enables diatoms to be particularly resistant to decay, so they may persist in an environment for a long time. It is their abundance, uniqueness and resistance that has allowed diatoms to be of such great use in the field of forensic limnology, that is the study of freshwater ecology in a legal context.

So how can these minute microorganisms help determine the circumstances surrounding a suspicious death?

Imagine a person drowning. As the head is submerged, water is inhaled into the lungs, along with any microorganisms contained in that water. In this case, diatoms. So the presence of diatoms in the lungs proves death by drowning? Not at all. Water can passively reach the lungs regardless of whether the victim was dead or alive by the time they reached the water. However if the victim is alive, when diatoms hosted by the water reach the lungs, they will be circulated around the body via the bloodstream, being deposited in different bodily tissues and internal organs.

So with this in mind, the investigator may be able to conclude that cause of death is likely to have been drowning if these diatoms are detected in the internal organs. At this point it is necessary to note that diatoms may already exist within the body, as these algal communities are found in various environments other than water. It is therefore necessary to establish a kind of match between the diatoms in the suspected drowning medium and those inside the body of the deceased. By studying the species of diatoms present and their abundance, it may be possible to conclude whether the diatom populations are consistent with one another. Interestingly diatom populations can also vary seasonally, thus may be able to provide some insight into the time of year in which a victim drowned based on the diatoms extracted from the remains. Comparisons such as these can be made by collecting water samples and extracting diatoms from bodily tissues and internal organs (often through acid extraction), before comparing the diatoms using light or electron microscopy.

The possible applications of the study of diatoms is by no means limited to these scenarios.

Numerous features of diatoms make these microorganisms an ideal focus for analysis in forensic investigations. Their minute size means that they can be readily transferred from the crime scene by objects or people, with perpetrators unlikely to be aware of the presence of these organisms. The resilience brought about by the silica-based cell wall allows for them to persist in the human body even beyond later stages of decomposition, during which time cause of death by pathological means may be more difficult. The distinctive morphology of diatoms allows for species to be distinguished from one another, and their abundance and variation results in different bodies of water developing very distinctive assemblages of diatoms.

Unfortunately the use of diatoms as an indicator of cause of death by drowning is somewhat controversial, highlighting the need for further research in this area of study.

References

Horton, B. P. Boreham, S. Hillier, C. The development and application of a diatom-based quantitative reconstruction technique in forensic science. 2006. University of Pennsylvania Scholarly Commons.

Krstic, S. Duma, A. Janevska, B. Levkov, Z. Nikolova, K. Noveska, M. Diatoms in forensic expertise of drowning – a Macedonian experience. For Sci Int. 127(2002), pp. 198-203.

Pollanen, M. S. Diatoms and homicide. For Sci Int. 91(1998), pp. 29-34.

Scott, R. S. Morgan, R. M. Jones, V. J. Cameron, N. G. The transferability of diatoms to clothing and the methods appropriate for their collection and analysis in forensic geoscience. 241(2014), pp. 127-137.

Featured Image – https://www.flickr.com/photos/carolinabio/6622267417

April 30, 2015 by Locard's Lab

Geochemistry and Clandestine Graves

Perpetrators of fatal crimes will on occasion attempt to conceal their wrongdoings by burying the evidence – that is, attempting to bury human cadavers. This can be problematic during a forensic investigation for a number of reasons. Firstly, the search for a victim’s body may well be relatively blind, with investigators having little or no idea as to where a body has been buried. In some instances, a body may well be so damaged or decomposed that little recognisable human remains are present. The perpetrator may later remove the body from the burial site, perhaps fearing discovery, leaving behind no obvious trace that a body was ever buried there.

So what can investigators do to determine if an area of soil was the site of a clandestine grave (illicit burial site)? A number of methods that have been developed to tackle this question.

Certain chemical compounds may be indicative of decomposing flesh. Sterols have been suggested as a potential biomarker for decomposition fluids – that is, the presence of them in soil could indicate whether or not a body has decomposed in that location, depending on the types of sterols present and in what amounts. Sterols are a class of organic compound, of which cholesterol is perhaps the most well-known sterol present in animal cells. This compound can be found in plants too, but in a significantly smaller amount, thus the unexpected presence of cholesterol in soil will typically indicate some kind of animal-related activity. Research examining the decomposition fluids in soils found sterols to be beneficial in this application (Von der Luhe et al, 2013). A number of pig carcasses were buried over a few months, with soil samples being collected from underneath the cadavers at different time points after burial. Cholesterol and coprostanol were detected in the soil, and it is these substances that were of particular interest to the researchers. Coprostanol is formed via hydrogenation of cholesterol in the intestinal tract of higher mammals, thus it is considered a useful biomarker associated with the faecal matter of animals such as humans and pigs. The concentration of these compounds was greater during the time period in which the pigs were undergoing the putrefaction stage of decomposition, at which point fluids would be leeching into the soil. This suggests a certain time frame in which these compounds are useful as indicators of decomposition fluids.

Cholesterol

The research suggested that, as the cadaver decomposes, decomposition fluids leak into the soil, depositing cholesterol and coprostanol (and a whole range of other substances). Thus the presence of these compounds in a particular area of soil, particularly if nearby similar areas did not contain them, could indicate previous decomposition of a human (or equally a pig or other animal) in the area. However it is vital to note that these compounds could equally be detected in the soil as a result of faecal matter, though potentially in considerably lower concentrations than those produced by a whole decomposing body.

Other compounds resulting from decomposition are of equal interest in detecting potential gravesites. Adipocere, also known as grave wax, is an insoluble, white substance known to form if a body decomposes in very specific conditions. The presence of this substance in soil can of course indicate the decomposition of a body, but how does one distinguish between the decomposition fluids of a human and those of another mammal? Research has aimed to answer this question using isotopes (Bull et al, 2009). By focusing on the ratio of ¹³C to ¹²C content of particular fatty acids from the fats of various animals, it was suggested that it is possible to distinguish between adipose fats from humans and those from other animals, such as pigs, though further work may be required to develop this application.

Other researchers are applying existing forensic techniques in a novel manner to the detection of clandestine graves. When the body decomposes, a significant amount of nitrogen is released, typically in the form of ammonium and nitrate (Hopkins et al, 2000). Ninhydrin, a compound already readily available to law enforcement due to its use as a method of fingerprint development, can produce a blue or purple pigment upon reaction with certain nitrogen-containing compounds.

Ninhydrin is typically used for visualising fingerprints (http://daten.didaktikchemie.uni-bayreuth.de)

One particular study examining ninhydrin reactive nitrogen (Carter at al, 2008) left a number of mammalian cadavers to decay over a period of a month, after which soil samples from the burial sites were collected and analysed for ninhydrin reactive nitrogen. This work discovered that cadaver burial resulted in the concentration of NRN in the soil approximately doubling, thus concluding that it may be possible to use ninhydrin as a presumptive test for gravesoil. Of course this particular method is somewhat limited by the fact that any mammalian cadaver (and plants or faeces for that matter) will most likely produce this increase in nitrogen-containing compounds which will react with ninhydrin, but an interesting application of an existing indicator nonetheless.

The various methods of using the chemical analysis of soil to detect clandestine graves are plentiful and fascinating. Despite the limitations, namely the possibility of animal faeces and non-human decomposition providing false positive results, these techniques may at the very least act as a kind of presumptive or complimentary test for possible burial sites.

References

Von der Luhe, B. Dawson, L. A. Mayes, R. W. Forbes, S. L. Fiedler, S. Investigation of sterols as potential biomarkers for the detection of pig (S. s. domesticus) decomposition fluid in soils. Forensic Sci Int. 230 (2013), pp. 68-73.

Bull, I. D. Berstan, R. Vass, A. Evershed, R. P. Identification of a disinterred grave by molecular and stable isotope analysis. Sci Justice. 49 (2009), pp. 142-149.

Carter, D. O. Yellowless, D. Tibbett, M. Using ninhydrin to detect gravesoil. J Forensic Sci. 53 (2008), pp. 397-400.

April 23, 2015 by Locard's Lab

Identifying Insects with Spectroscopy

Entomology, that is the study of insects, can provide vital information during a forensic investigation. After an individual dies their body begins to undergo a complex decomposition process almost immediately, attracting a variety of insects along the way who wish to colonise, feed on the temptingly putrefying remains and reproduce.

Specialists have been taking advantage of this fact for hundreds of years, allowing us to discover that the types of insects present on a cadaver and the age of these insects can prove invaluable in estimating how much time has passed since the victim died (known as the post-mortem interval). Simply put, certain species prefer the decomposing corpse at different stages in the decay process, and with the right information, investigators can study the insects and their ages and begin to develop a kind of timeline.

https://www.flickr.com/photos/sharman/6331634

Currently, accurately identifying species and establishing the development stage of an insect can be time-consuming and requires the expertise of an entomologist and potentially DNA analysis. This is obviously not ideal – your average police force does not have an entomologist on hand, nor do they have oodles of times to dedicate to insect identification. Even with the assistance of an entomologist, accurately determining the age of maggots can be problematic. Although larvae may be of a certain age, their length and weight can be affected by a variety of factors that may not be accounted for, such as starvation (Singh and Bala, 2009).

As you might expect, researchers are searching for ways to resolve this issue, and analytical chemistry might just be the answer.

As analytical chemistry progresses and increasingly advanced analytical techniques are developed, we are seeing more and more fascinating applications of these instruments to established areas of study. In a recent study published in Forensic Science International, researchers took a well-established technique and applied it to forensic entomology. In this case, they used a form of infrared spectroscopy.

Infrared spectroscopy is an analytical technique which determines the amount of radiation absorbed by a molecule. Infrared light is directed towards to sample and, depending on the molecule, a certain amount of radiation will pass through the sample and some will be absorbed. When a molecule absorbs radiation, the bonds within it begin to vibrate. Different bonds will vibrate and be influenced by surrounding atoms to a different extent, thus allowing for a unique ‘spectrum’ to be produced. This spectrum is essentially a graph displaying how much radiation was absorbed by the sample at what wavelength. Scrutinising this spectrum can allow the analyst to determine what kind of molecules are present. Although this is not sufficient to specifically identify compounds, the spectrum produced can at least be used to distinguish between different samples, which will produce different spectra. The spectra essentially act as ‘fingerprints’ for different substances.

Typical IR spectroscopy spectra.

If you want to know a bit more about this technique, Compound Chemistry has a great little page on IR spectroscopy.

So back to how this analytical technique can be useful in forensic entomology. The proof of principle study to which I’m specifically referring aimed to both identify the species of larvae and the life cycle stage using vibrational spectroscopy, in this case Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. A slightly long-winded name, but in short this is simply a form of IR spectroscopy that allows in situ analysis of solid or liquid samples without the need for sample preparation. Anyone who has spent many painful hours preparing samples for analysis will appreciate the benefit of this.

Three species commonly encountered at incident scenes were used in the study; C. vomitoria, L. sericata, and M. domestica (that is the bluebottle fly, the green bottle fly and the common housefly respectively). One of these species (the C. vomitoria) was also selected for a study focussing on the life cycle, in which spectra were collected for each time point in the insect’s life cycle. Scans were based on a crushed mixture of epidermis and internal matter (not possible for a ‘no maggots were harmed during the making of’ notice then). The results were promising, indicating FTIR spectroscopy could be a great tool in forensic entomology.

A portable FTIR: http://www.chem.agilent.com

But surely there is a whole range of analytical instruments out there (yes, there sure is), so why would this one be any more suitable for forensic entomology? One of the major benefits of FTIR is the possibility of handheld IR instrumentation, which basically means it can be used in situ at the scene of a crime or other incident. Granted the investigator would need the appropriate equipment, but it beats shipping samples back to the lab and waiting for analysis. IR spectroscopy is a non-destructive technique (okay, the insects were somewhat mutilated in this study, but nevertheless the samples themselves remained after analysis). The ability to perform analyses without destroying the sample has a huge benefit, particularly if the available sample is limited, allowing for alternative tests and future analysis to be conducted if necessary. This of course is an advantage in forensic science. Also of great benefit to a legal investigation, IR instrumentation is fast, with spectra being collected in a matter of minutes.

There is however the glaring problem of the cost of analytical instrumentation. As I previously stated, your average police force may not have a forensic entomologist on hand… they equally may not have the funds to purchase analytical instrumentation such as IR spectrometers.

Bearing in mind this was merely a pilot study, using a very limited sample size, the research shows some promising results – that it is possible to classify species and life cycle stage using IR spectroscopy. Were this to be expanded upon, you could theoretically develop a database of IR spectra collected from different species of insects at different stages of development, allowing future spectra obtained from unknowns to be compared and, hopefully, identified.

References

Pickering, C. L. Hands, J. R. Fullwod, L. M. Smith, J. A. Baker, M. J. Rapid discrimination of maggots utilising ATR-FTIR spectroscopy. Forensic Sci Int. 249 (2015), pp 189-196.

Singh, D. Bala, M. The effect of starvation on the larval behaviour of two forensically important species of blow flies (Diptera: Calliphoridae). Forensic Sci Int. 193 (2009), pp. 118-121.

March 26, 2015 by Locard's Lab

The Challenges of Forensic Air Crash Investigations

The subject of aircraft crashes has unfortunately dominated the media headlines at numerous points over the past couple of years, ranging from malicious attacks, such as the plane shot down over Ukraine in July 2014, to still somewhat unresolved incidents, in particular the missing Malaysia MH370 flight that disappeared in the ocean in March that same year.

As one would expect, the investigation that ensues is often gruelling, time-consuming and expensive, requiring the expertise from a variety of fields and often collaboration between countries in accordance with International Civil Aviation Organization guidelines. The investigation of any incident scene, whether criminal, accidental or natural, will be accompanied by its own set of difficulties and problems. The forensic investigation of a plane crash is certainly no exception.

The Investigation

The discovery of an incident involving an aircraft typically comes to light when air traffic control loses communication with the plane, particularly if the pilots had previously made a distress call. The exact location of the crash site may or may not be immediately obvious. Either way, a number of teams will be instantly gathered to begin the investigation, with the earlier hours of the incident being the most crucial.

As much information about the aircraft will be gathered as possible, particularly pertaining to its maintenance history and if there had been any signs of a potential fault, as well as details of the crew. Weather information will be collected and scrutinised for any possible occurrences that may have placed the aircraft in difficulty. If there were any witnesses to the incident, such as people in the area in which the plane crashed, they may be able to provide beneficial information regarding how the plane looked prior to crashing.

The investigation of the scene itself will involve a tremendous amount of documentation, as with any crime or accident scene, though made immensely more difficult by the likely sheer size of the area under examination. Examination of the wreckage itself may, in theory, aid in establishing the cause of an aircraft incident, such as through examination of a faulty component or the discovery of debris that would suggest a malicious attack. For instance, in the investigation of the shot-down MH17 plane, there was evidence on the outside of the aircraft indicating something trying to get into the plane (in this case the projectile used to destroy it), as oppose to anything originating inside the aircraft. Unfortunately the nature of such incidents are often so catastrophic that even locating such remains are a challenge, as more often than not the debris is strewn over a vast area. Even if components are retrieved, they may have been destroyed by the crash or fire, reducing their value as evidence.

Crash Location

The ease of the investigation of the wreckage and recovery of bodies is largely determined by the site of the crash. When an incident occurs at sea, simply locating the wreckage is the first greatest challenge, and in many cases the plane and thus the people on board are never found. If the wreckage is located, the process of recovering victims and airplane components from the bottom of the ocean is by no means an easy task. This can often be a dangerous process involving highly-skilled divers and specialist equipment, such as inflatable balloons which can be attached to pieces of the aircraft to draw them to the surface for collection.

Should the aircraft strike land, depending on the nature of the crash the wreckage will most likely to scattered across a vast area, making simply identifying and controlling the entire incident scene a great difficulty, let alone effectively investigating it. In the case of the March 2015 Germanwings crash, the incident occurred in the Alps, a mountainous area making scene investigation especially troublesome and dangerous for those involved.

Aviation incidents can occur over any country, which in itself can be problematic in terms of managing the investigation. As in the case of the aircraft shot down over Ukraine in 2014, pieces of the wreckage remained in dangerous areas riddled with war and fighting. These factors can make the vital immediate access to the crash site difficult if not impossible, greatly hindering an investigation. There may be further complications brought about by explosions and fires caused when the aircraft crashes, making accessing the wreckage more difficult as well as potentially compromising already damaged evidence.

Data Recorders

When an aviation incident takes place, one of the primary goals of investigators is finding a device known as the black box (though these are actually bright orange in colour). Modern aircraft are now equipped with a number of data recording devices, well-built components designed to record as much data as possible relevant to the aircraft prior to and during the incident and ideally survive crashes and fires. The black box is a small device generally bolted to the aircraft’s tail so as to prevent severe damage in the event of a head-on crash.

These devices are also equipped with an underwater locator beacon, which is activated if the recorder comes into contact with water. This allows investigators to detect ‘pings’ emitted by the device so that the black box can be located. However the device can of course only function for a limited amount of time, typically for around 30 days before battery life is exhausted, so investigators are working against the clock to locate this vital piece of equipment.

The flight data recorder is a device used to record certain operating parameters from the aircraft’s system, the examination of which may be able to indicate if there were any major faults with the aircraft. This can record a range of details including altitude, time, direction, and airspeed, as well as the movement of certain aircraft components, auto-pilot details and fuel levels. The cockpit voice recorder is a unit which typically records sound from microphones worn by the pilot and the co-pilot as well as from the cockpit area. If this device can be recovered intact (or at least intact enough to retrieve data files), documentation of what was audibly occurring in the cockpit prior to and during the incident can be invaluable, particularly if the pilots were unable to communication with air traffic control at the time.

Victim Identification

Looking at the catastrophic scenes caused by some aircraft crashes, it is of no surprise that identification of passengers and crew can be an difficult task. Locating the bodies of victims can be a gruelling enough mission, particularly if the incident occurred at sea or over a poorly-accessible area, such as the recent incident over the Alps. When victims are located, the bodies may be damaged beyond recognition by the crash itself, fire, water, or even decomposition if recovery of bodies was delayed. In these instances specialised forensic experts may be required to carry out DNA analysis or examination of dental records, guided by the details known about the victims from flight manifests and the assistance of family members.

Lessons Learned

Despite a number of recent high-profile plane crashes, aviation remains one of the safest forms of transport, in part down to the strict operating and safety procedures adhered to, many of which were developed following aviation incidents and investigations. If investigators can at least in part deduce what happened to cause an incident, steps can be taken to prevent reoccurrence and make flying safer.

References

Anthiniotis, N et al. Scientific analysis methods applied to an investigation of an aircraft accident. Eng Fail Anal. 17 (2010), pp 83-91.

BBC News. Malaysia plane; Why black boxes can’t always provide the answers. [online] Available: http://www.bbc.co.uk/news/magazine-26721975

Robson Forensic. Airplane Accident Investigations. [online] Available: http://www.robsonforensic.com/practice-areas/airplane-accident-investigation-expert