March 26, 2020 by Locard's Lab

Maggot Analysis with Mass Spectrometry

A new proof-of-concept study by researchers at the University at Albany in New York has developed a mass spectrometry-based technique for the rapid species prediction of blow fly larvae for use in forensic investigations.

Entomological evidence (evidence relating to insects) has proven invaluable to forensic investigations for decades, particularly in the estimation of time since death. Insects which feed on decomposing remains, known as necrophagous insects, will colonise a body in a reasonably predictable pattern, with different insects arriving at different stages throughout the decomposition process. Different species of flies, beetles and mites are commonly encountered. Blow flies in particular will often arrive at the scene within minutes of death to lay eggs on the body. As these eggs hatch, larvae (or maggots) emerge to feed on the decomposing remains. By studying the type and age of insects present at a scene, it may be possible to estimate the time since death, or postmortem interval.

The ability to achieve this hinges on the correct identification of insect species, which is unfortunately not always straightforward. The larvae of different species of blow fly are visually very similar, thus difficult to distinguish by eye. For this reason, maggots are often reared to maturity for species identification, with adult blow flies exhibiting more distinguishing physical differences. Inevitably the rearing of maggots to adulthood is a time-consuming process that requires the expertise of a forensic entomologist.

In recent years, researchers have tried to develop more rapid approaches to insect species identification, particularly using chemical analysis. Researchers at the University at Albany in New York have been applying direct analysis in real time mass spectrometry (DART-MS) to the analysis of insect evidence to provide a rapid species identification tool. In DART-MS, the sample is placed between the DART ion source and the inlet of the mass spectrometer, allowing chemical components in the sample to be ionised and drawn into the MS for direct analysis. DART-MS requires minimal or no sample preparation and results can be obtained almost instantly. Using this technique, Rabi Musah and her team have already demonstrated the ability to determine the species of larvae, pupae and adult flies, highlighting a promising new tool in rapid species identification in forensic entomology.

However, until now this research has focused on the analysis of individual species. In a real-world scenario, maggots present on the body may consist of multiple different species, therefore any techniques developed for rapid species identification of larvae must be able to work with mixed samples. In a recent study, the team have taken the method one step further by examining the potential to identify larvae from mixed species.

Blow flies of various species were collected from Manhattan, New York. Maggots were submerged in 70% ethanol and the solution exposed to the ion source of the DART-MS to produce chemical signatures of both individual species and combinations of species. Mixtures of two, three, four, fix and six different species were analysed. Using the chemical profiles produced, a predictive model was constructed for the subsequent identification of unknown insect samples. Using this model, maggot species could be established with an accuracy of up to 94% and a confidence interval of 80-95%. Individual insect species are readily differentiated, with different species producing distinct chemical profiles. Similarly, mixtures of two different species could also be differentiated. As might be expected, samples containing a higher number of species were more difficult to differentiate.

Although only a proof-of-concept study and further validation is required, the study demonstrates that DART-MS could offer a way of rapidly determining the species of blowfly larvae, thus allowing investigators to establish which insects are present at the scene of a death and work out postmortem interval faster.

Beyramysoltan, S. Ventura, M. I. Rosati, J. Y. Giffen, J. E. Musah, R. A. Identification of the Species Constituents of Maggot Populations Feeding on Decomposing Remains—Facilitation of the Determination of Post Mortem Interval and Time Since Tissue Infestation through Application of Machine Learning and Direct Analysis in Real Time-Mass Spectrometry. Analytical Chem, 2020, In Press.

May 29, 2019 by Locard's Lab

The Smell of Death: Confirming Decomposition using Volatiles in the Air

Odour mortis, or the ‘smell of death’, refers to the chemicals released from the body during decomposition. Renowned forensic anthropologist Arpad Vass, who has studied the chemical changes occurring in the body after death for many years, recently shared the details of a particularly interesting scenario. The article, published in the May 2019 issue of Forensic Science International, details a fascinating case in which the occurrence of human decomposition was demonstrated based solely on chemical compounds in the air for the first time, without any human remains actually being found at the scene. The article doesn’t specify suspect or victim details, but anyone familiar with the case will recognise it instantly.

First, a brief introduction. In 2008, a woman was charged with the murder of her daughter, allegedly storing the victim’s body in the boot of her car for several days before disposing of the remains and dumping the car. Police had initially been alerted to the incident by the suspect’s parents, who had picked up their daughter’s abandoned car and noticed a foul decomposition-like odour coming from the vehicle. Coupled with the fact they had not seen their granddaughter in several weeks, the suspect’s mother promptly called 911.

The police soon took possession of the car and agreed that the scent of decomposition was emanating from the vehicle. Numerous cadaver dogs, specifically trained to detect odours from decaying bodies, alerted to the back of the car, further suggesting some kind of decomposing remains had been stored in the boot of the car. Fly pupae were also discovered. Entomological evidence is frequently associated with decomposing human remains, with flies and various other insects known to visit corpses to feed or lay eggs. Although no human remains were found in the car, several weeks later the body of the missing girl was found in a wooded area near the suspect’s home, and the case promptly turned into a murder investigation, with the victim’s mother as the prime suspect. However with minimal physical evidence linking the body to the suspect’s car, law enforcement turned to a somewhat unconventional tool to aid their investigation.

Various pieces of evidence were recovered from the vehicle, including segments of carpet, scrapings from the tyre wells, and various pieces of rubbish found in the car. Interestingly, investigators also collected some air samples from the boot of the car. Air can be sampled from remote locations using a technique that utilises air pumps to draw in gaseous analytes from the environment and capture them in a sorbent trap. This collection of trapped compounds can then be transported to a laboratory for analysis. In this case, about 35L of air was collected from the vehicle into a type of sorbent tube, then analysed using gas chromatography/mass spectrometry (GC/MS). GC/MS is a well-established analytical technique, allowing scientists to separate the individual chemicals in a mixture and identify those components. You can read more about how mass spectrometry works here.

This process allowed researchers to figure out exactly which volatile chemicals were present in the suspect’s vehicle and establish whether these are everyday compounds likely to be found in a car, or if they had some other source.

In the years leading up to this case a great deal of research had been conducted at the University of Tennessee’s Anthropological Research Facility. At this facility researchers were investigating, among other things, the odours produced during the decomposition of a human body. The odours created during this process are the result of volatile compounds produced as the body decomposes, and research has demonstrated that hundreds of individual chemical components are formed during this complex process. As part of research at the university, researchers had constructed a vast database of hundreds of chemicals detected during the process of human decomposition, including the different decomposition stages at which those chemicals appear. By comparing the chemicals detected in the vehicle with those stored in the database, it was possible to identify compounds known to be produced during the decomposition process. There was an 80% match between the compounds detected in the boot of the car and those chemicals considered to be relevant to human decomposition. Furthermore, unusually high levels of chloroform were also detected in the boot of the car.

The results from the air samples collected and chemical extracts from various other artefacts in the car led the researcher to conclude that there was a very high likelihood of a decomposition event occurring in the boot of the car. Many of the compounds detected in the vehicle could only be logically explained by the presence of decomposing remains.

Despite these findings (and various other pieces of evidence presented in court), the jury reached a verdict of not guilty for the charge of murder. Not too surprising an outcome, considering the use of air analysis to detect decomposition had not previously been used in a legal investigation. However in closing arguments, the defence stated that the victim had in fact been placed in the vehicle for transport (claiming the victim’s death had been accidental), ultimately confirming the results of the analysis.

Vass, A. A. Death is in the air: confirmation of decomposition without a corpse. Forensic Sci. Int. (2019). doi:10.1016/j.forsciint.2019.05.005

Vass, A. A. Odor mortis. Forensic Sci. Int. 222, 234–241 (2012).

December 3, 2018 by Locard's Lab

Interview with Forensic Taphonomist Professor Shari Forbes

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

Forensic taphonomist Professor Shari Forbes.

I am a Canada 150 Research Chair in Forensic Thanatology and the Director of the Secure Site for Research in Thanatology (SSRT). The SSRT represents the first human taphonomy facility in Canada and is the only place in this distinct climate where we can study the process of human decomposition through body donation. My role is to lead and conduct research at this facility, specifically in the field of forensic thanatology and decomposition chemistry. This role also involves engaging collaboratively with our external partners who can benefit from the research and training we conduct at the facility, notably police, forensic services, search and rescue teams, military, human rights organisations, and anyone involved in death investigations.

What initially attracted you to your particular field of research?

I have always had a passion for science and knew that I wanted to pursue a career in a scientific field where I could clearly see the impact of my work. When I was in high school, I enjoyed reading crime novels and probably understood what forensic science entailed better than most people (this was before the advent of CSI, Bones, NCIS, etc.!). My love of science combined with my interest in criminal investigations naturally led to pursuing a career in this field. At the time, there was only one university in Australia that offered a forensic science degree so the decision of where and what to study was relatively easy. Although chemistry wasn’t my strongest subject at school, I enjoyed the degree because it applied chemistry to forensic science and in this way, I could understand how my skills would help police investigations.

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

My research focuses on the chemical processes of soft tissue decomposition and the by-products released into the environment. This can include compounds released into air, water, soil, textiles, or anything surrounding the body. The majority of my research at the moment focuses on the release of volatile organic compounds (VOCs) into the air to better understand the composition of decomposition odour. Although this is not pleasant work, it is very important to understand the key compounds used by cadaver-detection dogs for locating human remains. If we can identify the key VOCs and determine when they are present, we can enhance the training and success of cadaver-detection dogs in complex environments such as mass disasters.

You were heavily involved in the establishment of the Australian Facility for Taphonomic Experimental Research. What were some of the greatest challenges in this and how has the facility since developed?

It took approximately 3.5 years to establish AFTER from the day we started planning it to the day it opened in January 2016. I have since realised this is not that long compared to some of the other facilities that are currently operating but there were challenges and hurdles that we faced along the way. In Australia, establishing a human taphonomy facility essentially requires three things: 1) an organisation willing to lead and support it; 2) a body donation program; and 3) accessible land that can be used for taphonomic research. We were fortunate that the University of Technology Sydney (UTS) had these three things and we also had the financial and in-kind support of all of our partners including academic institutions, police services and forensic laboratories. Once we had this support and made the decision to proceed, we still needed to seek approval from our local council to use the land for this purpose; apply for funding to build the facility; and apply to have the facility licenced to hold human remains for the purposes of taphonomic research and training. Thankfully, everyone we engaged with was highly supportive of the facility and willing to work with us to ensure we followed all legislation and regulations. We also ensured we had a strong communication plan to raise awareness with the general public about the benefits of these facilities and how important the research is to assist in the resolution of death investigations.

The Australian Facility for Taphonomic Experimental Research

Since opening, we have been amazed by the general interest in AFTER and the number of people wanting to donate their body. We have also increased our partnerships to benefit more police and forensic services as well as others services such as the cemetery industry. We are currently planning to provide more training opportunities, particularly relating to disaster victim recovery and identification, and to establish more AFTER facilities across Australia to better represent the diverse climates experienced across the country.

You recently left the University of Technology Sydney to relocate to Canada. How will your role and research be changing as you make this move?

I was honored to be the Director of AFTER and it was a difficult decision to leave Australia. However, I recognise the importance of these facilities and the need to establish them in other countries so when I was asked to open Canada’s first human taphonomy facility, it was an opportunity I could not turn down. My experience in Australia has already assisted greatly in establishing the facility in Quebec and we will certainly be able to open the facility much more rapidly as a result. Like in Australia, we hope it acts as a template for future facilities across Canada since this country also has very diverse climates. In reality, neither my role nor my research will change significantly. The greatest change will be the climate and its impact on the process of decomposition!

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

It sounds like a cliché, but I always encourage students to pursue a career in a field they are passionate about. If you had told me 20 years ago that I would being leading not one, but two ‘body farms’ I would never have believed it (especially after just reading Patricia Cornwell’s novel that gave these facilities that name!). But I knew I was passionate about studying a science that was deeply applied and had a clear impact on society. I had no idea where it would lead me or even if I would get a job in the field, but without that passion, I would not have been motivated to do any of the things I have done; namely: complete my degree, continue on with a PhD, do research in decomposition chemistry, and ultimately become an academic so that I could continue my passion of conducting forensic taphonomy research. So if you are going to do something for the next fifty years, make sure it is something you love doing!

Find out more on the Secure Site for Research in Thanatology website.

This is Part 17 of our series of interviews with forensic professionals. If you’re a forensic scientist (academic or industry) or a crime scene investigator and would like to be part of this series of interviews, get in touch by emailing locardslabblog[at]gmail.com.

July 6, 2017 by Locard's Lab

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.

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

June 22, 2016 by Locard's Lab

Interview with Forensic Anthropologist Dr Anna Williams

hud pic

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

I am Principal Enterprise Fellow (equivalent to Reader or Associate Professor) in Forensic Anthropology at the University of Huddersfield. My time is divided between teaching undergraduate and postgraduate students, supervising MSc and PhD students, and doing research and forensic casework. I teach on the BSc/MSci Forensic and Analytical Sciences, and the MSc in Forensic Anthropology and the MSc in Risk, Disaster and Environmental Management. Part of my role is also to engage with the public and communicate our research to lay people, including school children, interested adults and other scientists. I regularly present at academic conferences, local interest groups, Science Festivals and public events. This year, I am presenting at the Royal Society Summer Science Exhibition. I have also been featured in several TV science documentaries, and regularly consult for TV shows like Bones, Rosewood and Silent Witness. I also write a blog about my adventures in forensic anthropology.

What initially attracted you to this field of work?

I did a mixture of sciences and humanities at A Level and could never decide which I liked best, so I chose Archaeology and Anthropology as my first degree. There, I was fascinated by what you could tell about individuals by their skeletal remains, for example about hominid evolution. Then I discovered the burgeoning science of Forensic Anthropology, on a short course at Bradford University, and that was it, I was hooked! I love how you can glean all sorts of information from the smallest pieces of evidence. I have always loved logic problems, and forensic anthropology offers the most exciting puzzles. The fact that it is often confronting, challenging and disturbing, and could help to bring criminals to justice, just serves to add to its appeal for me.

Can you tell us about the research you are currently involved in at the University of Huddersfield?

I specialise in decomposition and taphonomy (the study of how bodies decay in different environments). To do this, I use an outdoor decomposition laboratory. I lead a research group currently doing research into the gases given off by decomposing cadavers (we use pigs that have died of natural causes), and comparing that to the efficiency of police dogs that are specially trained to find dead bodies. We’re also looking at how skin colour changes in surface or water environments, and trying to find ways to improve our estimation of post-mortem interval and post-mortem submersion interval. Other research is focussed on the taphonomic changes that occur to bone and teeth in hot, arid environments. I am also running a citizen science project in order to improve age estimation of unknown individuals from dental eruption. There is a webpage and online questionnaire for anyone who would like to help us build a large, modern set of tooth eruption data to see if dental eruption ages are changing.

Aside from research, are you often involved in police casework, and what does this typically involve?

Sometimes I am asked by the police to attend crime scenes or mortuaries to undertake forensic examination of decomposed or skeletonised remains. They can be either the victims of crime, or the remains of people who have gone missing. I will determine whether they are human or animal, and if they are human, I will estimate the age at death, sex, stature and ancestry of the individual(s), and try to say something about their lifestyle, disease, injury and how they died. I work in conjunction with forensic archaeologists and odontologists, as well as pathologists, to reach an identification. I also do consultancy for forensic science providers and, on occasion, a mass disaster company that helps to ‘clean up’ after disaster and repatriate the victims. I am involved in disaster victim identification and the Emergency Operations Centre.

The existence of so-called ‘body farms’ has sparked great interest in the media. Are there plans to establish such a facility in the UK? What are the primary challenges associated with this?

I believe that Human Taphonomy Facilities, or ‘Body Farms’ as they have become colloquially known, are vital for the advancement of forensic science. We owe all that we know already about human decomposition to the Forensic Anthropology Center at the University of Tennessee, and there is so much more to learn. We need to know how human conditions like diabetes, cancer, smoking and drug use affect our decomposition, which is something we cannot learn from experimenting with dead pigs. Unfortunately, a lot of the data generated by the ‘Body Farms’ in the USA and Australia are not directly relevant to forensic cases in the UK or Europe, because of the different climate, insects and scavengers. The UK is falling behind the USA and Australia by not having one of these outdoor laboratories where vital decomposition research can be done on donated cadavers. There was an attempt to establish a Body Farm in the UK in 2011, but this failed for a variety of financial and political reasons. I think the main obstacles to getting one set up in the UK are lack of funding, public awareness and rivalry between academic institutions. I hope that, in the near future, we will be able to create a facility where researchers, academics and practitioners will be able to work together to improve methods of search and recovery, post-mortem interval estimation and identification of human remains.

Do you have any words of advice for students wishing to pursue a career in forensic anthropology?

Forensic anthropology is a very competitive field, and there aren’t many jobs out there, so you need to be dedicated and determined. It can also be hard work and distressing, so decide carefully whether you want to pursue a career in it. The best way to make yourself stand out from the crowd of other applicants to jobs is to have experience, so try to get as much hands-on experience as you can. This doesn’t have to be forensic (although, of course, that would be preferable), but can be in archaeological units or museums or hospitals (or even zoos), somewhere where you can deal with human (or animal) bodies.

Images from Research

These pictures show the progression of decomposition in a small (10kg) pig. The first picture shows the pig in the fresh stage, when post-mortem interval was less than 24 hours. The second picture shows the pig in the active decay stage, 25 days later. The brown froth is decomposition fluid that has been agitated by the movement of maggots. The body was bloated with decomposition gases, but has now collapsed, and the intestines are escaping. The skin has desiccated, but the hair is still intact. The skin has darkened and become leathery in texture. The bones are becoming detached from the body.

Surface pig 1, day 0

Surface pig 1, day 25

Website: www.forensicanna.com

Twitter: @Bonegella

Also, you can follow #scentofdeath and #teamtaphonomy

November 26, 2015 by Locard's Lab

Interview with Forensic Entomologist Susan Gruner

SG2

What is your expertise within forensic science and what does this type of job role typically involve?

Forensic entomology can be divided into three groups: stored-product, urban and medico-legal (or medico-criminal) entomology. I specialize in the field of medicolegal forensic entomology. Generally speaking, forensic entomologists can estimate a postmortem interval (PMI) or time since death based upon the presence (or lack thereof) of insects collected on or near a body. Certain families of insects arrive in a predictable manner on corpses, especially the calliphorid flies (blow flies). Based upon the ages of specimens collected, a PMI can be estimated. Age of the specimens is directly related to the temperatures that the insects are experiencing; the higher the temperature (obviously to a certain limit), the faster they grow.

In a best-case scenario, when a body is found, a forensic entomologist should be called and he or she would collect and process the insect evidence. The second best scenario would be that properly trained law enforcement personnel would collect the insect evidence and then send it to a forensic entomologist for processing.

On occasion, forensic entomologists have to testify in court, just as would any other expert witness. The testimony usually focuses on time of death, but not always. Sometimes a body may have been moved and the calliphorids present can sometimes help determine such a thing. There was a case where a woman was murdered in a parking lot in the city of Jacksonville (Duval County). Her body was dumped in rural Clay County, but not before those city flies had a chance to deposit eggs on her body. The trial was held in Duval County.

Calliphorid female flies are attracted to areas of trauma on a body, where they will deposit eggs. Sometimes the trauma is not noticeable to the naked eye, but can be determined during autopsy. But forensic entomologists know that flies congregate and deposit eggs on areas of trauma on a body.

In September 2006, I received a call from the Jacksonville Sheriff’s Office that the body of an elderly man had been found in a culvert off of I-10, a major highway. I did not know what a culvert was until this case (for those who do not know, it is a tunnel that carries water under a road). It was necessary to climb down a steep angled wall to make an entomological collection. When I reached the body, I noticed that the man had hundreds of calliphorid larvae around his neck despite a lack of visible wounds or bruised skin. I told the detectives that the medical examiner would find the cause of death to be strangulation. I got a lot of strange looks from the detectives that day, but the autopsy showed the man had indeed been strangled to death.

I guess you could say that being a forensic entomologist requires getting very dirty, too. The first time I ever returned from a death scene, my husband had to have the car detailed because it smelled like human decomposition. But as horrific as it sounds, I continue to be fascinated by what calliphorid larvae can reveal about death and decomposition and how they can help solve crimes.

What led you to become involved in forensic entomology?

I thought I wanted to be a veterinarian, and one of the ways to get into vet school is to major in entomology. On the first day of my first entomology class, the professor handed out an article about the Body Farm in Tennessee. I changed my mind about wanting to be a veterinarian that instant.

What in particular was the focus of your PhD research in forensic entomology?

The main focus of my doctoral research was to study the life cycle of the forensically important calliphorid, Chrysomya megacephala (the oriental latrine fly). The main focus of my research was to study their development rates at different ambient temperatures.

Historically, calliphorid development rates have been studied by placing a few hundred calliphorid eggs in a cup (or cups) in a rearing chamber set to a certain temperature. The time is monitored as the larvae grow through three larval stages, a puparial stage, and then emerge as adults.

But the problem with testing in this manner is that calliphorid masses generate heat. Even a small amount of maggots can generate heat above ambient, thus the set chamber temperature is not really the temperature that the growing larvae/pupae are experiencing. For the development research, I used 10 larvae per container; an amount not capable of generating heat above ambient temperature.

Liver is a standard feeding substrate for calliphorid larvae in colony. But the small amount of liver needed for only 10 larvae per Petri dish dried up quickly, killing the larvae, so I developed a liver agar feeding substrate that would not dry up¹. Then I was able to study precisely how the larvae developed at eight different temperatures.

This was grueling work in which larvae were checked at 4-hour intervals 24/7 for most of the duration of the development research, which took 15 months. As I had no funding to hire anyone to help me, I trained my husband to do it. He took the day shift, going back and forth to the lab every 4 hours as I tried to get some sleep. I spent nights (from 9 PM to 9 AM) in the lab. I was so sleep-deprived that it would have been dangerous for me to drive back and forth during the middle of the night.

There were times when we had to examine every larva individually to determine stage. This required looking at the anal spiracles of a larva under a microscope with a gentle but steady hand. During all those months of research when we were sleep-deprived and miserable, we never squished or killed a larva. I am very proud of that.

I also studied different size maggot masses and their respective temperatures as they grew in age, size and volume. And finally, I studied different models that could potentially be used to calculate age of a larva (or larvae). Most forensic entomologists use a linear model to estimate a PMI, but insect development is not linear, especially that of calliphorids.

Do you believe there are any common misconceptions surrounding this field of work?

There are too many to list, but I will name a few.

It is nothing even remotely similar to anything seen on TV.
Sadly, I know of only one person who is a full-time forensic entomologist in the USA (in Texas). Most who received their M.S. and PhDs studying forensic entomology are not teaching or practicing forensic entomology. They may take a case or testify in court on occasion, but that is rare.
Despite the potential importance of collecting insect evidence at death scenes, it is not done. The vast majority of detectives, crime scene technicians, etc., have no idea how to collect insect evidence and forensic entomologists rarely get called to process the insect evidence at death scenes.

Do you think that there are any significant gaps in research in forensic entomology?

Yes. I would say the biggest problem in this field is the poor quality of research, especially when it comes to studies regarding development rates of forensically important insects. Most (almost 80%) of the manuscripts published in peer-reviewed journals in the past 30 years lack replication and have poor experimental design. This is an embarrassment to the science and needs to change. But, there are many dedicated scientists in this field who are trying to do good work.

Finally, how do you feel about the extent to which forensic entomology is harnessed in legal investigations?

Forensic entomology is overlooked much of the time. Collection of insect evidence is not difficult, but as I mentioned above, such evidence is rarely–if ever–collected.

Twitter: https://twitter.com/Entomophila

If you’re a forensic scientist (academic or industry) or a crime scene investigator and would like to be part of this series of interviews, get in touch by emailing locardslabblog[at]gmail.com.

This is Part 6 of our series of interviews with forensic professionals.

Interview Series Part 1 – Interview with Forensic Identification Scientist Alexandre Beaudoin

Interview Series Part 2 – Interview with Forensic Expert Robert Green OBE

Interview Series Part 3 – Interview with Forensic Expert & Consultant Gareth Bryon

Interview Series Part 4 – Interview with Forensic Identification Specialist Donna Brandelli

Interview Series Part 5 – Interview with Forensic Video Analyst David Spreadborough

Interview Series Part 6 – Interview with Forensic Accountant Sundaraparipurnan Narayanan

May 21, 2015 by Locard's Lab

From Mummies to Grave Wax – The Preservation of Human Cadavers

Warning: Graphic images included.

When we envisage the decomposition of a corpse, the images that probably come to mind are of a rapidly-decayed, foul-smelling body quickly turning to sludge and bones. But there are actually other pathways a body can take following death, some of which can be of great importance in a forensic investigation. I’m namely talking about mummification and adipocere.

Mummification

Thanks to the Ancient Egyptians, we all know about mummification, a particular process of body preservation. When these guys mummified their dead, this routine typically involved the removal of the internal organs, most notably the brain, which was pulled out through the nostrils. Finally, the body would be wrapped in linens and salt and left to dry. The end result would be a remarkably well-preserved body displaying features that would have usually been lost to decomposition.

Aside from this especially famous post-mortem ritual, there are actually a number of ways by which a body can become mummified. For instance, more modern intentional mummification would utilise chemicals to preserve the body. But how can mummification occur naturally, and what are the implications of this in a forensic investigation?

The natural preservation of a cadaver is highly dependent on the surrounding environment, with only very specific conditions causing the body to mummify. A range of factors can play a part in this phenomenon, including temperature, humidity and the action of bacteria and other microorganisms.

In hot, dry climates moisture can evaporate from the skin at such an accelerated rate that the process of mummification can occur. As the skin is rapidly dehydrated, it often takes on a dark and leathery appearance. The internal organs may be preserved to an extent, though will typically undergo some level of decomposition so are at least likely to be smaller in size. Hot, dry climates can also hinder bacterial growth, limiting the bacterial decay and further preserving the body. Hot, sandy deserts are perhaps amongst the first scenarios that come to mind when considering mummification, but mummified remains have also be discovered in attics, basements, and even within the walls of buildings.

Conversely, especially cold and dry environments can also bring about mummification. The cold temperatures can significantly slow microorganism activity, once again reducing the rate of decomposition and aiding in preservation. A famous example of this is the natural mummy Otzi the Ice Man, believed to have died thousands of years ago but preserved through mummification induced by extremely cold temperatures.

Mummifying conditions are not limited to temperature-based factors. Environments of extreme salinity (salt content) can preserve cadavers. The majority of bacteria cannot survive in highly salty conditions, thus severely reducing microbial action on the body. Furthermore salt itself acts as a desiccant on the soft tissue, dehydrating the body and drawing out water much as high temperatures would. An example of this type of mummification was experienced in Iran, where a number of mummies were found in the Chehrabad salt mines.

Mummified remains have also been found in bogs or marshland, in which the excess water, organic material and anaerobic (oxygen-free) environment prevents a great deal of bacterial action, thus preserving the body. This is something of a contrast to the typical hot, arid conditions mostly associated with mummified remains, but bogs with particularly acidic water, low temperatures and a lack of oxygen can essentially pickle a body. Thousands of these “bog bodies” have been recovered over the years, perhaps the most famous being the Lindow Man, determined to be the victim of a prehistoric ritual killing.

The conditions described may not necessarily induce mummification throughout the entire cadaver, but in some cases may cause localised mummification, if only particular areas of the body are exposed to these conditions. Mummification most often occurs in the face, scalp, chest and back, but typically begins in the extremities such as the fingers and toes.

Adipocere

Another phenomenon that can assist in preservation of a cadaver is the formation of adipocere. Known as “grave wax”, this is a soapy, white or grey wax composed primarily of saturated fatty acids such as palmitic and stearic acid formed through the hydrolysis and hydrogentation of body fats (Forbes et al, 2005). Numerous theories have been put forward to suggest how adipocere forms, namely the saponification, hydrogenation and fat migration theories.

A cadaver presenting adipocere. Credit: Kumar et al, 2009

The type of environment required for adipocere formation is somewhat different from that suitable for mummification. It is often encountered in especially humid graves with little or no air access, thus oxygen-poor, such as a bog or certain bodies of water. The formation of adipocere can take weeks if not years to form depending on the climatic conditions, the rate at which it forms being further affected by the environment and circumstances surrounding the cadaver. Depending on the extent to which it forms, adipocere can produce a waxy layer across the body and act as a barrier against the usual process of decomposition, providing significant protection over time as adipocere itself is fairly resistant to decay.

So these are some of the alternative routes of decay a body can take post-mortem. But what does this mean to the forensic scientist?

In some cases, the occurrence of mummification or adipocere formation can be of assistance to a forensic investigation, as it may be possible that certain aspects of the deceased person’s appearance and even any injuries they might have acquired will be preserved. Mummified tissues can even be rehydrated to aid in visualising injuries and other distinguishing marks. Similarly, the formation of adipocere can preserve tissues and organs along with recognisable facial characteristics. This can in theory aid in identification if the victim is unknown or even determining cause of death.

Furthermore, just as the stage of decomposition of a body can roughly indicate the post-mortem interval (time since death), mummification and adipocere can provide some indication in that a certain amount of time is required for mummification to occur. Approximately 6-12 months are required for the natural mummification of an adult, with a child’s body requiring less time to become mummified (Gitto et al, 2015), though in some cases mummification has been reported in a matter of weeks or even days (Sledzik and Micozzi, 1997). Of course these time periods can vary widely depending on climatic conditions and a number of other factors, but they may provide assistance nonetheless. To an extent it may be possible to determine the rough age of the remains based on the weight of the mummified cadaver, as more recent bodies will be heavier than those which are older and have lost a greater proportion of water content.

So given the right conditions, processes such as mummification and adipocere formation can interestingly be a great aid to the forensic investigator.

References

Bereuter, M. T. L. Lorbeer, E. Reiter, C. Seidler, H. Unterdorfer, H. Post-morten alterations of human lipids – part I: evaluation of adipocere formation and mummification by desiccation. Human Mummies. 3 (1996), pp. 265-273.

Bryd, J. H. Castner, J. L. 2010. Forensic Entomology: The Utility of Arthropods in Legal Investigations. Boca Raton, Florida: CRC Press.

Forbes, S. L. Bent, B. B. Stuart, H. The effect of soil type on adipocere formation. For Sci Int. 154 (2005), pp. 35-43.

Gitto, L. Bonaccorso, L. Maiese, A. dell’Aquila, M. Arena, V. Bolino, G. A scream from the past: A multidisciplinary approach in a concealment of a corpse found mummified. Journal of Forensic and Legal Medicine. 32 (2015), pp. 53-58.

Kumar, T. S. M. et al. Early adipocere formation: A case report and review of literature. Journal of Forensic and Legal Medicine. 16 (2009), pp. 475-477.

Rich, J. Dead, D. E. Powers, R. H. 2005. Forensic Medicine of the Lower Extremity. Totowa, New Jersey: Humana Press Inc.

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.