Instant Insect Identification to Aid Forensic Entomology Investigations

Instant Insect Identification to Aid Forensic Entomology Investigations

During the investigation of a suspicious death, entomological (that is, insect-related) evidence may be able to provide vital clues as to when the victim died. Determining time since death, or post-mortem interval, can be one of the most important aspects of such an investigation, so it comes as no surprise that a great deal of research has been directed towards improving these estimations.

Insects can play a huge role in estimating time since death. Various types of species of insect will often visit the scene of a death in a relatively predictive manner, either to feed on the decomposing remains (known as necrophagous insects), to prey on other insects present, or to find a suitable place to lay their eggs. Blow flies, a group which includes common flies such as the bluebottle and the greenbottle, are often of particular interest. Forensic entomologists will typically study the insects, eggs and larvae present at a death scene, utilising the type of bugs found and their stage of development to track back to the likely time at which they arrived, thus when the victim may have died. However in order to accurately do this, entomologists must often collect insect specimens for closer inspection and even to rear to adulthood in order to determine the exact species, which is evidently a time-consuming process requiring a high level of expert knowledge.

For the first time, researchers at the University of Albany have applied a technique called direct analysis in real time with high resolution mass spectrometry, or DART-HRMS for short, to the analysis of blow fly eggs. Published in the latest issue of the journal Analytical Chemistry, the technique has demonstrated the possibility of almost instantly differentiating between different fly species based on the amino acid profiles of the eggs.

DART-MS, developed in 2005 by Dr Chip Cody of JEOL, is an ambient ionisation mass spectrometry technique that allows for samples to be directly analysed without any time-consuming sample preparation steps, and perhaps most importantly without destroying the sample. The sample is simply presented in its native state between the ion source and the inlet of the mass spectrometer, enabling compounds present in the sample to be ionised and drawn into the instrument for analysis and identification.

dartms

Sampling interface of DART-MS. Source: Wikimedia Commons

During this investigation, researchers used pieces of pork liver to attract a number of different blow fly species before transporting them to the laboratory. The flies were reared until they lay new eggs, which would be the focus of the analysis. The study utilised specimens of a number of species, including Calliphora vicinia, Lucilia coeruleiviridis, Lucilia sericata, Phormia regina, along with specimens from the Phoridae and Sarcophagidae families. Even to the eye of an expert, the eggs of these specimens are often indistinguishable. The eggs were simply placed in an ethanol solution and the mixtures directly subjected to DART-HRMS analysis.

The technique focused on the analysis and identification of amino acids in the eggs, essentially enabling researchers to produce a chemical fingerprint unique to eggs of a particular species. Examination of the mass spectra showed that the different species exhibited a unique chemical fingerprint, and by using multivariate analysis it was possible to better visualise the similarities and differences between amino acids detected in the eggs of different species.

Unsurprisingly, many amino acids were common to multiple species. For instance, alanine, isoleucine and proline were detected in four of the species, whereas valine was detected in all but one of the egg samples. However some compounds were unique to particular species, and it is these unique amino acids that will prove to be most beneficial in differentiating between the eggs of different species. For instance, glutamine and tryptophan were only present in the eggs belonging to P. regina. Interestingly, the research also demonstrated the ability to distinguish between families as well as species, with some compounds only detected in the eggs of specific families.

By using this particular technique, almost instantaneous identification could be achieved. Of course this research has included only a very limited number of species, thus a much bigger investigation would be necessary before the technique would really be beneficial to a legal investigation. Not only would further species need to be included, but another potential development would be the production of a chemical profile database against which unknown insect samples could be compared. Developed further, the use of DART-MS could save investigators a lot of time in the identification of insects of forensic interest.

 

References

Cody, R. B., Laramée, J. A. & Durst, H. D. Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions. Anal. Chem. 77, 2297–2302 (2005).

Giffen, J. E., Rosati, J. Y., Longo, C. M. & Musah, R. A. Species Identification of Necrophagous Insect Eggs Based on Amino Acid Profile Differences Revealed by Direct Analysis in Real Time-High Resolution Mass Spectrometry. Anal. Chem. (2017) In Press

 

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Interview with Forensic Physician Samar Abdel azim Ahmed

samar

What is your professional background in forensic science?

I am an associate professor of Forensic Medicine in Ainshams University Faculty of Medicine in Egypt. I received my doctorate degree 10 years ago with honours from ASU and then proceeded to work on my educational capacity. I studied for a second Masters degree from Maastricht University and Suez Canal University in Health professions education. I then received a scholarship from ECFMG in USA for a fellowship program in Health professions education in FAIMER, Philadelphia.

What is your current job role and what does this work involve?

Currently I teach forensic Medicine to fourth year medical students together with my administrative job as the director of the Centre of Excellence in Forensic Psychiatric research. This centre is a product of a Newton Mosharafa Fund that I received from the British council and the Science Technology Development fund in Egypt to establish forensic psychiatry research trends in Egypt. At the moment I am working on establishing partnerships within the scope of forensic psychiatric service improvement.

What initially attracted you to this field of work?

I am a physician by training but I was attracted to the field of forensics mainly challenged by the importance of the service that one can offer to justice by giving a voice to the voiceless. My work as a forensic physician is mainly to advocate for those who are victimized and to prevent further injustice by uncovering the truth that can only be seen by forensics.

Can you tell us about the research you are currently involved in?

At the moment my point of focus is forensic psychiatric patients. I am indulged in studying the service offered in my country with the hope that I can import state of the art practices from the UK utilizing the cooperation agreement that I have set with them. The first part of the study is mapping the patient’s body in Egypt with special reference to the determinants of the length of their stay in the high secure wards. This requires a lot of work to establish a culture and understanding of predictors of violent behaviour. This work comes within my funded project that we have now come to call LIFE project.

Why is this work important to the field of forensic science and what do you hope to achieve by carrying out this research?

Our hope is to be able to establish guidelines to predict violent patient behaviours and thus be able to predict patients who are in need of extended stay in forensic wards. This will help in turn to reduce unnecessary length of stay of patients. By the end of this work I hope to be able to publish a white paper of effective forensic psychiatric practice as a guiding document to help in the decision making process when patients are discharged.

Do you have any words of advice for students wishing to pursue a career in your field of work?

My advice for students who want to pursue a career in forensic medicine is to specialize as early as possible. The earlier you specialize and maybe even subspecialize the quicker you grow in the field. Master your passion area and own it then try to build on it from early on. You build your name from day one in the field so build a name that goes with a specialization. It is also important to understand why you are in the field. Understand that you give bones a voice and that without you the truth will be buried indefinitely so it is important to take this calling very seriously.

Keeping the Skies Safe with Analytical Chemistry

Ever since events such as 9/11, the Lockerbie bombing and the (fortunately) failed shoe bomber, the stringency of airport security has been ever increasing. Anyone who has passed through an airport has no doubt witnessed the occasional swabbing of luggage or electronic items. The staff will take a quick swab of the item, stick it into a mysterious machine and usually send the passenger on their way with little explanation of what has just occurred.

But what exactly are they testing for in this scenario, and just what is the instrument they’re using?

As one might expect, the biggest target of this security step is explosive substances as a counter-terrorism measure, in addition to illicit narcotics in an attempt to crack down on drug trafficking. In an airport setting, the analytical testing technique of choice is ion mobility spectrometry.

Ion mobility spectrometry (IMS) is an analytical technique used to identify chemical compounds based on the differences in the movement of ions under an electric field. The concept for the technique was established in the early twentieth century, however it was not until the 1970s that the instrumentation was actually properly developed. There are currently tens of thousands of IMS devices deployed around the world. Not only are they utilised in airports for drug and explosives screening, but also by the military for the detection of chemical warfare agents and in industrial settings to monitor air quality. The range of applications is potentially vast, but the principles of operation are the same.

As you may have witnessed, a small swab is rubbed over the surface to be tested, typically a piece of luggage or an electronic device such as a laptop, before being inserted into the ion mobility spectrometer. As the sample needs to be introduced in its gaseous form, the swab may be subjected to heating in order to thermally desorb analytes from the swab and allow them to be transported into the instrument for analysis. In order to manipulate the analytes entering the instrument, they must first be converted into ions, their charged form. Ionisation is typically achieved using a radioactive source, such as 63Ni (nickel-63) or 241Am (Americium-241), which first form reactant ion species from the carrier gas (usually air), which then leads to the ionisation of the sample material. These newly-formed ions will then enter a region under an electric field and drift towards a series of electrodes. The ions will pass through the drift region at different speeds depending on the shape and size of the ion clusters and strike the electrodes, the signals being amplified and detected. Depending on the instrument and needs of the analysis, either positive or negative ions will be produced (in some cases both simultaneously).

ims

IMS schematic. Source: Smiths Detection (www.smithsdetection.com)

The IMS utilised in airports will typically hold a database of known explosive and narcotic substances against which to compare samples. There will be a certain threshold, typically based on peak intensity, that must be reached before a positive identification will be indicated, and if there is a “match”, the operator will be alerted to a potential identification.

In comparison to other analytical tools available, ion mobility spectrometers are far from being the best. For instance mass spectrometry, an alternative technique for the analysis and identification of chemical compounds, can offer greater sensitivity, higher resolution, improved accuracy and better identification. So why use IMS? It essentially comes down to cost and ease of use. The simple design and ability to operate at atmospheric pressure means the instruments can be fairly small in size, some even being hand-held and so rendering them completely portable. They have low power consumption, so can simply be powered by a few AA batteries. The ease of use of the IMS means anyone can be trained to use the instrument, thus technical or scientific expertise is not required.

But what is perhaps most important for use in an airport setting with potentially thousands of passengers each hour, is the ability to conduct analyses quickly, and this is something that the IMS can offer. Many commercial ion mobility-based instruments can provide results in a matter of seconds. For instance, the IONSCAN by Barringer (now owned by Smiths Detection) boasts the ability to detect over 40 explosives and narcotics in just 8 seconds.

In a security setting there are three primary types of IMS that may be encountered. The smallest of the devices are handheld and sample by drawing in analytes present in the atmosphere. These may be used to analyse potential hazards relating to unattended baggage, for example. The second type, which is perhaps the most commonly encountered IMS in airports, is a benchtop instrument which requires introduction of the sample via some type of swab. And finally, some airport security units may utilise a larger, human-sized IMS portal. This setup uses airflow to dislodge particles of explosives or drugs from clothing or the passenger’s body and analyse them.

Unsurprisingly, the instruments are not infallible, and false positive or negative results are a possibility. Some ions will have the same drift time so may be indistinguishable from known explosives or drugs, triggering an alarm. In actual fact this response may simply have been caused by a cosmetic or pharmaceutical product that happens to produce a response similar to a known narcotic. On the contrary, dirt, oil and other contaminants may mask the presence of substances of interest, thus causing no alert despite the presence of a drug or explosive.

Furthermore, the IMS is somewhat limited in that it can only identify the presence of a compound contained within its database. So whereas it may be able to detect common explosives such as RDX, TNT and PETN, and frequently encountered narcotics such as cocaine, heroin and cannabis, it would not necessarily alert to the presence of an unknown compound (unless it was very similar in chemical structure to something in the database).

Fortunately research in the field of analytical chemistry is constantly ongoing, aiming to improve instrumentation and analytical techniques to resolve these issues and ultimately produce more reliable and robust security measures.

 

References

G. Ewing et al. A critical review of ion mobility spectrometry for the detection of explosive and explosive related compounds. Talanta. 54 (2001) 515-529.

Homeland Security Science & Technology. IMS-Based Trace Explosives Detectors for First Responders. [online] Available: https://www.dhs.gov/sites/default/files/publications/IMSTraceExploDetect-SUM_0506-508.pdf

Smiths Detection. Ion Mobility Spectrometry (IMS). [online] Available: https://www.smithsdetection.com/index.php?option=com_k2&view=item&layout=item&id=40&Itemid=638

VX and Other Deadly Nerve Agents

It has now been confirmed that Kim Jong-nam, the half-brother of North Korean Leader Kim Jong-un, may have been assassinated using a highly toxic nerve agent known as VX. The attack occurred last week (13th February) in Kuala Lumpur airport, suspected to have been committed by two women who reportedly sprayed the chemical into his face before fleeing the scene.

VX, or S-[2-(Diisopropylamino)ethyl] methylphosphonothioate, is a nerve agent initially developed at the Porton Down Chemical Weapons Research Centre in Wiltshire, UK in 1952. Having originally been the focus of research elsewhere into the development of new organophosphate compounds as pesticides, the British military soon established an interest in the compound and continued its development.

vx-wiki

S-[2-(diisopropylamino)ethyl] methylphosphonothioate) or VX

Typically encountered in liquid form, this clear or sometimes amber-coloured, oily substance is notoriously difficult to detect, lacking in both taste and odour. Its toxicity makes it one of the deadliest chemical warfare agents, requiring as little as 10mg adsorbed through the skin to be fatal. Its deadliness is only further increased by the persistence of the agent, making it difficult to decontaminate people and areas tainted with the chemical.

Mechanism

The mechanism of action of VX is identical to many similar nerve agents. The compound can enter the body by a range of potential routes, including ingestion, inhalation or skin contact. Once inside the body, VX inhibits the function of acetylcholinesterase (AChE), an enzyme responsible for catalysing the breakdown of acetylcholine. Acetylcholine is released over a synapse following an electric nerve impulse, ultimately resulting in a muscle contraction. However when VX binds to the active site of acetylcholinesterase, it renders the enzyme inactive, thus preventing it from breaking down the acetylcholine. As the nervous system is flooded with excess acetylcholine,  repeated muscle contractions occur, eventually resulting in asphyxiation due to constant contraction of the diaphragm muscle.

The effects of VX will typically occur immediately after exposure, beginning with coughing, shortness of breath and a tightness in the chest. A headache and blurred vision soon follows, along with symptoms such as vomiting, diarrhoea and abdominal pains. Given a sufficient dose, seizures will then occur as the drug attacks the nervous system, eventually resulting in a coma and asphyxiation.

If administered promptly, there are antidotes for VX. Atropine, typically administered by injection, is an anti-nerve agent that blocks the acetylcholine receptors, alleviating the symptoms brought on by the nerve agent. However it is worth noting that compounds such as atropine are toxic in their own right and, although they may save the person’s life by alleviating the effects of the nerve agent, they will still have an adverse effect on the patient. In addition to this, pralidoxime (or 2-PAM), can be administered to reactivate the enzyme, thus reversing the effects of VX. 2-PAM is a safer compound to use than atropine, but its effects are much slower.

Other Nerve Agents

VX is just one of many known toxic nerve agents. Nerve agents can typically be classed as either G-series or V-series. G-series agents were first synthesised by German scientists during World War II, and include tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF). The first compound to be discovered, tabun, was accidentally synthesised by Dr Gebhardt Schraeder, who was investigating the development of organophosphate-based pesticides. The German army soon realised the potential use of such compounds, and went on to fund the development of other nerve agents such as sarin. The G-series chemicals are all clear, colourless liquids at room temperature, but are largely utilised as gases due to their high volatility.

The V-series nerve agents, which include VX, VE, VG, VM and VR, were developed a few years later, initially in the UK but some later in Russia. Unlike the G-series compounds, V-agents are very persistent and are not easily washed away or degraded, meaning they can remain on surfaces for long periods of time.

Fortunately the V-series nerve agents have generally not been exploited outside of military research, and the death of Kim Jong-nam may well be the first known use of the toxic agent in an assassination. However the G-series have received a great deal of malicious use and attention over the years, ranging from the Tokyo sarin subway attack in 1995 to its recent use in the Syrian civil war

VX, along with numerous other toxic nerve agents, were banned under the Chemical Weapons Convention of 1993, rendering the manufacture, possession and use of such substances illegal.

 

References

BBC News. VX nerve agent: The chemical that may have killed Kim Jong-nam. [online] Available: http://www.bbc.co.uk/news/world-asia-39073558

University of Bristol Chemistry on the Screen. VX Nerve Gas. [online] Available: http://www.chm.bris.ac.uk/webprojects2006/Macgee/Web%20Project/nerve_gas.htm

 

 

 

 

 

Interview with President of IsoForensics Inc., Lesley Chesson

southern-utahDifferent forms of elements–called isotopes–are found everywhere in the environment. These isotopes are incorporated into materials in varying ratios and the abundances of different isotopes thus serve as a record of the material’s formation. Analysis of a material for its distinct isotope signature can subsequently be used to reveal its history. Investigators have applied stable isotope analysis to a variety of materials of forensic interest including drugs, explosives, money, food, ivory, and human remains. For example, the isotopes in human hair protein can reveal the age of an individual, what s/he ate, and even how often (and where) s/he travelled.

What is your professional background and how did you come to be involved in IsoForensics?

I have a master’s degree in biology, with a lot of microbiology and chemistry experience. I entered the world of isotope forensics when I was hired to raise Bacillus subtilis (a cousin of the anthrax bacterium) under a variety of conditions – in liquid media, on agar plates, with different nutrients, etc. Because organisms record information about the environment in the isotopes of their tissues, the goal of this project was to develop models that allowed investigators to predict the growth conditions of a dangerous bacterium–such as anthrax–from its isotopic characteristics.

From its start in academia, IsoForensics developed into a private analytical services and research firm that explored novel forensic applications of isotope analysis. I enjoyed the challenges offered by that exploration. Since my first work on the B. subtilis project, I’ve been involved in other projects on human remains, foods and beverages, illicit drugs, and explosives.

Tell us about the work IsoForensics is involved in and what kind of clients do you typically work with?

Currently, IsoForensics provides a lot of human remains testing in unidentified decedent cases. The goal is to use the isotope records contained in hair, nails, bone, and teeth to reconstruct the travel history of individuals and provide new evidence on their origins: Were they local to the area of discovery? Had they traveled prior to death? Where might they have traveled? We work with a variety of law enforcement groups in this casework.

In addition to service work, we conduct basic and applied research through funded grants and contracts. One recent project has started to investigate the origins and ages of seized elephant ivory to understand the structure of illegal trade networks in Africa and Asia.

What are some of the most common sample types you are asked to analyse, and does anything pose a particular challenge?

In any given month, we can analyze a variety of materials – human and wildlife remains, illicit drugs, explosives, etc. One of the most challenging measurements we make is for strontium isotope ratios. There is so little strontium contained in organic materials that prep work takes place in clean lab settings. The preparation of materials for radiocarbon dating is also challenging since we must be extremely careful about contamination of “old” materials with “modern” carbon. However, these challenges are worthwhile since strontium isotopes can provide potentially useful geolocation data about materials while radiocarbon dating provides quantitative information on the “age” of materials.

Are there any areas of isotopic analysis that could benefit from further research and development?

Yes. Isotope forensics benefits from better and better models/methods for interpreting data. It’s one thing to compare isotope measurement results from sample to sample or from sample to a reference databank, but it’s another thing altogether to understand the process(es) driving isotopic variation in materials. For example, are the results we observe due to differences in TNT manufacturing process? Or coca plant physiology? Or elephant diet?

Isotope analytical techniques also change over time as better instrumentation is developed. Understanding the impact of different analysis techniques on measured isotope ratios is extremely important when comparisons are made – especially in legal settings. A major focus of the field is the standardization of practices and protocols, to generate comparable results over time and space (e.g., from lab to lab).

How has the need for isotope analysis in forensic investigations changed over the years, if at all?

The forensic application of isotope analysis has been increasing the past 10-20 years. This is partly due to changes in analytical techniques, which have made isotope ratio measurements faster and cheaper. In addition, those who could benefit most from forensic isotope data–law enforcement, regulators, etc.–have become more aware of the technique and it potential usefulness in various types of investigations. We as forensic scientists and isotope analysts can do even more to spread awareness about the technique and its many applications.

Finally, do you have any advice for students hoping to pursue a career in this field of work?

Isotope analysis is one (extremely useful!) tool in a forensic scientist’s toolbox. Having a background and training in other areas–such as anthropology, analytical chemistry, biology, biochemistry, geology, law, or statistics–can be very important when applying isotope analysis techniques and interpreting the resultant data. The field of isotope forensics is relatively small compared to some other forensic disciplines, so be sure to read papers, attend meetings, and network with scientists working in the field.

Visit the Isoforensics Inc. website for more information.

Sex Determination with Raman Spectroscopy

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%.

malefemaleramanspectra

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.

 

Forensic Failures: Ray Krone & Bite Mark Blunders

Forensic Failures: Ray Krone & Bite Mark Blunders

In 2002, Ray Krone became the 100th wrongfully imprisoned person to be exonerated by DNA analysis in the US, but only after spending ten years of his life detained in Arizona prisons, including a number of years on death row.

On the morning of 29th December 1991, the owner of the CBS Lounge in Phoenix, Arizona went to his bar to discover the door unlocked, the lights on, and the naked body of 36-year-old Kim Ancona on the floor of the men’s bathroom. The victim, who had worked in the bar as a waitress, had been brutally stabbed to death.

The subsequent examination of the scene was somewhat fruitless and, although saliva was recovered from Kim’s body, little other physical evidence could be found. The only piece of evidence investigators had to work with was a series of bite marks found on the victim’s breast and neck. As the investigation continued, police began interviewing those close to the victim in attempts to shed light on the events leading up to her death. It transpired that the victim had told a friend that Ray Krone, a regular customer at the bar, was to help her close up the bar the previous night. Investigators jumped at the possibility of a potential suspect.

During an interview with Krone, a detective noticed that he had a very distinctive deformity of his front teeth, no doubt causing a unique bite mark. This characteristic would later lead to the nickname of ‘the Snaggletooth Killer’. Krone was happy to oblige when asked to provide a Styrofoam impression of his teeth for comparison purposes.

However unfortunately for him, a forensic odontologist soon declared that the Styrofoam impression matched the bite marks found on the victim’s body.

Krone maintained his innocence, insisting that he was at home in bed at the time of the murder, a story corroborated by his roommate. Despite this, Ray Krone was arrested and charged with kidnapping, sexual assault and murder.

As the trial commenced, it was clear there was little evidence for the prosecution to present, so they focused their efforts on the bite mark comparison. They hired forensic odontologist Raymond Rawson to conduct the comparison between the bite marks on the victim’s body and the impression of Krone’s teeth. Using compelling video footage attempting to show the physical match between the two, Rawson informed the jury that the match was “100 per cent”, and that only Krone could have made those bite marks. The defence chose not to call upon their own court-appointed forensic odontologist.

Despite a lack of DNA analysis or eyewitness testimony linking Krone to the crime, and the bite mark comparison being the only physical evidence implicating him, Ray Krone was found guilty and sentenced to death.

Three years later Krone was awarded a re-trial due to the prosecution team concealing the persuasive video tape concerning the bite mark evidence until a day before the original trial, but once again he was found guilty. Despite the opportunity to rectify the conviction being wasted, trial judge James McDougall aired his uncertainty: “the court is left with a residual or lingering doubt about the clear identity of the killer. This is one of those cases that will haunt me for the rest of my life, wondering whether I have done the right thing”.

Forensic Odontology & Bite Mark Comparison

As DNA analysis was not carried out during this investigation, Krone’s conviction was almost entirely based on the ‘expert’ opinion that his teeth matched the bite marks on the victim’s body.

Dental identification is based on the theory that every individual’s dentition is unique, and thus bite marks made by a person will be distinguishable. In theory, this is true – we all have different combinations of jaw sizes, varying dental work and unique wear patterns to our teeth. Bite mark comparison may involve a variety of methods, including overlaying appropriately-sized photographs of teeth and bite marks and fitting together physical moulds.

At the time, there was little reason to doubt the testimony of the forensic odontologist hired by the prosecution. Raymond Rawson was a well-established expert who was certified by the American Board of Forensic Odontology, his findings in this case were supported by another expert, and the discipline of bite mark comparison had been practiced for almost 20 years. Furthermore, a 1984 study had provided “statistical evidence for the individuality of human dentition”. The expert witness testimony seemed perfectly reputable.

Well, that is until we look a little closer.

The seemingly convincing 1984 study was actually research conducted by Rawson himself, and has since been widely criticised as being a flawed study, largely because he used hand-traced dental impressions for his comparisons, a non-randomised subject selection process, and statistical tests not relevant to his type of data. Other experts had quite rightfully stated that the results of the study should absolutely not be used in a legal case.

A study conducted ten years previously comparing bite marks in wax and pig skin to the teeth of subjects stated that, although bite marks in wax were easily assessed, those made in pig skin were difficult to examine and the results unreliable. The research concluded that incorrect identification of bite marks on pig skin were made 24% of the time under laboratory conditions, and even as high as 91% of the time when based on photographs taken 24 hours after the bite marks were initially made. The study highlights the clear difficulties in subjective fields of work such as forensic odontology. Experts will often be required to examine bite marks that are hours or even days old, obscured by bruising and abrasions and typically not entirely representative of the biter’s teeth. At times it is challenging enough to merely identify an injury as a bite mark, let alone successfully compare it to a set of teeth.

Despite these apparent shortcomings, Ray Krone was to spend a decade of his life behind bars.

Exoneration

Fortunately for Krone, he had an undeterred family behind him maintaining his innocence and the means of hiring proficient legal help and in 2002, with the help of attorney Alan Simpson, he successfully appealed.

DNA analysis had become, by this point in time, a well-established technique frequently utilised in criminal investigations. Analysis of bodily fluids recovered from the crime scene a decade earlier soon proved not only Krone’s innocence, but also the identity of Kim Ancona’s real killer. Kenneth Phillips, a man with a long history of repeated violent sex offenses, was serving time in prison for the sexual assault of a 7-year-old girl, but at the time of Kim’s murder was living a mere 600 yards from the scene of the crime. Despite his close proximity to the bar, his deviant history and the fact that he was at the time of the murder on probation for the assault of a neighbouring woman, Phillips was never considered a suspect.

On 8th April 2002, Ray Krone left prison a free man, the 100th person to be exonerated by DNA evidence. He would certainly not be the last.

Krone now lives in Tennessee, where he has since dedicated his time to criminal justice reform and the campaign for the abolition of the death penalty.

“I would not trust the state to execute a person for committing a crime against another person. I know how the system works” – Ray Krone.

 

References

Innocence Project. Ray Krone. [online] Available: http://www.innocenceproject.org/cases/ray-krone

New Scientist. Bite-mark evidence can leave false impression. [online] Available: https://www.newscientist.com/article/dn4758-bite-mark-evidence-can-leave-false-impression

Rawson, R. D. et al. Statistical evidence for the individuality of the human dentision. J For Sci. 29(1984), pp245-253.

State v. Krone, 897 P.2d 621, 182 Ariz. 319 (Ariz. 06/22/1995)

Whittaker, D. Dome laboratory studies on the accuracy of bitemark identification. Int Dent J. 25(1975) pp. 166-171.

Cover Image – https://www.flickr.com/photos/girlstyle/449883708/in/photostream