CSI: Crime Scene Insects and rapid analysis methods to aid the … · 2016-04-11 · CSI: Crime...

CSI: Crime Scene Insects and rapid analysis methods to aid the field of

Forensic Entomology

July to August 2012, USA

A report for the Winston Churchill Memorial

Trust

by

Hannah Moore BSc 2012 Fellow

Keele University, Staffordshire, UK

“Never give in-never, never, never, never, in nothing great or small, large or petty, never give in except to convictions of honour and good sense. Never yield to force; never yield to the apparently overwhelming

might of the enemy.”– Sir Winston Churchill

WCMT: Crime Scene Insects and rapid analysis to aid FE

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Acknowledgements

My first acknowledgment is of course to the wonderful Winston Churchill Memorial Trust

who’s funding and support made it all possible. I simply cannot thank them enough for

giving me such an amazing opportunity and I will forever be endeavored to them all and

their amazing work. I shall continue to promote the trust as best I can as it is the very least

I can do in return.

I would also like to say a huge thank you to Dr Andrew Hart (2005 fellow) for telling me

about the trust and urging me to apply. His advice through the application process and the

contacts he gave me over in the USA was invaluable.

I would like to acknowledge all of my fantastic hosts as a whole, as I individually

acknowledge them later in the report. They were all so welcoming and I couldn’t have

asked for more knowledgeable, fun and enthusiastic people to be around during my time in

the USA.

I would like to thank Keele University for allowing me to take time out of my PhD studies

to go to the USA and my PhD supervisor, Dr Falko Drijfhout for his support during my

trip as well as being my referee.

Lastly, I would like to thank Dr Martin Hall for kindly agreeing to be my second referee as

well as his ongoing support during my PhD studies. Forensic entomology was an area that

was very new to me and he has gone out of his way to guide and advise me.


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Forensic Entomology and Direct Analysis in Real Time

(DART) study in USA

Aims

The first aim of my Winston Churchill Fellowship was to gain more knowledge and hands

on experience in the area of forensic entomology. Forensic entomology is far more

accessible in the USA in comparison to the UK so being able to meet with leading experts

in the area and view case report as well as go into the field would give me invaluable

insight.

The second aim of my fellowship was to incorporate my current PhD studies with the

powerful analytical instrument, Direct Analysis in Real Time (DART) coupled to a Time of

Flight Mass Spectrometer (TOF-MS). I wanted to look at the hydrocarbons on the cuticle

of forensically important insects in the USA and see if the DART-MS could be used for

rapid species identification.

Introduction

My Background

I am in the final stages of a PhD at Keele University studying chemistry applied to forensic

entomology. I did not start my university life in the conventional way. Due to a love of

horse riding, I was not much for school when I was younger. I was juggling my GCSE’s

alongside training in Buckinghamshire with the junior British eventing squad. In my eyes,

this was going to be my career and school was just getting in my way! Needless to say by

the age of 18 I had a handful of weak GCSE’s and no A-levels to my name. I pursued my

horse riding dream of representing my country until my beloved horse suffered a serious

injury, abruptly ending my journey. It was then that I realised I needed a more secure

qualification. I had to practically beg the course director of the science foundation year at

Keele University to accept me, as I was under qualified. He compromised with a math’s test

to ensure I was capable of the level that would be required of me, and I have never looked


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back. I successfully achieved my BSc in Chemistry and Forensic Science and moved onto

my PhD. I have had many doors closed in my face and I have made it my objective to prove

every person behind the doors wrong. People are capable of great things if they are willing

to work at it. Life is not a bed of roses…

Forensic Entomology

Forensic entomology is a branch of forensic science that uses insects and their arthropod

relatives that inhabit decomposing human remains to aid legal investigation. Due to the

abundance of insects that inhabit decomposing remains, they offer a variety of forensic

evidence. In particular, the first inhibitors, known to be the blowfly, can provide

information ranging from an estimation of how long a person has been dead (Post Mortem

Interval), to determining whether the deceased had ingested drugs prior to death.

The minimum Post Mortem Interval (mPMI) is an estimation based on the time taken for

the developing insects to reach the stage they are at when first discovered on the corpse

and it has a start point and an end point. The start point is when the first fly lays its eggs

on the body and the end point is upon discovery of the body and identifying the

development stage of the oldest colonising species. A medical pathologist can usually give

an accurate PMI up to 72 hours after death depending on the state of the corpse and the

climate (the milder the conditions the faster the decomposition process). However, the


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longer the body is discovered after this window of time, the more inaccurate the PMI will

be which is when a forensic entomologist would be called to the crime scene.

Decomposition of human remains attracts insects that arrive on the corpse in waves known

as the succession pattern. The extent of the body’s decomposition will determine which

insects are present, as the body goes through different changes in temperature and

moisture making it hospitable for different insects at different times. For example, beetles

tend to be more attracted to the dry stage as they have mouth pieces which can bite flesh,

as well as preying on the fly larvae present, whereas flies are more attracted to the fresh

and bloated stage.

The female blowfly will usually lay in open wounds if there are any present or in the body’s

natural and moist orifices such as the ears and nose.


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The life cycle of the blowfly contain six stages (eggs, three larval stages, pupal and adult

fly). The three larval stages increase in size as they develop, known as 1st instar, 2nd instar

and 3rd instar. There is also another stage that occurs before pupation, known as the post-

feeding stage. This is where the larvae stop feeding and migrate away from the body

seeking cool, dark and damp conditions in order to pupate. Adult flies then eclose from the

puparial cases and the cycle continues.

When a forensic entomologist attends a scene, they will collect entomological evidence

present on and around the corpse. Some samples will be killed and preserved at the crime

scene for further analysis. Others will be kept alive and reared in the laboratory at the same

temperature and humidity as the crime scene. The overall aims are to firstly identify the

insect, preferably down to species level. This will allow for the correct developmental data

to be utilised, since different species develop at different rates. They will then try and

calculate the age of the larvae, achieved by looking at the size and weight and the use of

growth charts and temperature data. Larval growth is a function of temperature so the

ambient temperature at the scene will be taken, as well as maggot mass temperature and

meteorological data gathered from the closest station to where the body was found.

To gain access to anthropological research facilities as well as forensic entomology case

reports would give me a great insight into the field as this is something I currently have no

access to in the UK.

DART-MS

This instrument was developed in 2005 by Laramee and Cody and when coupled to high

resolution mass spectrometer, allows for rapid qualitative and quantitative analysis of a

wide variety of samples. DART’s potential is being exploited in a broad range of

applications from food science to drug analysis. DART offers rapid real time analysis with

the advantage of little to no sample preparation due to sampling from the surface of

numerous samples from tablets to blood. The sample can simply be held in front of the

helium stream and an instant mass spectrum is obtained. This can then be used to


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identify the compounds present in the sample. The classic example that co-inventor, Dr

Robert (Chip) Cody uses when demonstrating the capabilities of DART is waving a dollar

bills into the helium, causing a cocaine peak to appear instantly on the mass spectrum.

Dr Cody has also designed software allowing for the statistical analysis of Principal

Component Analysis (PCA) and Linear Discriminant Analysis (LDA) to be applied to

datasets obtained from DART spectra. This was utilised in my results obtained during my

visit with JEOL, USA (commercial market retailers of DART).

DART is been utilised widely in the USA in a variety of industries which are greatly

benefiting from its many capabilities. However, there are a handful of instruments in the

UK. My aim was to analyse insect samples collected from the USA to see if DART could

offer a rapid method of species identification. My overall aim would then be to promote the

work of DART back in the UK to try and get more companies to see the huge potential it

holds.

My Fellowship 2012

Itinerary

A) Texas: Dr Jeffery Tomberlin, Texas A&M University, College station, 6th July to 15th

July.

Dr Chip Cody with the DART


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B) Las Vegas: North American Forensic Entomology Association (NAFEA) conference,

16th July to 20th July

C) Michigan: Dr Richard Merritt, Michigan State University, East Lansing, 22nd July to

27nd July

D) Ohio: Dr Eric Benbow, Dayton University, 30th July to 2nd August

E) Ohio: Captain David Rose, Ohio State University Police, Columbus, 3rd August

F) Boston: Dr Robert (Chip) Cody, JEOL USA, Peabody, 6th August to 17th August


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Texas A&M University

I arrived in the US on 6th July to visit the

wonderful assistant professor, Dr Tomberlin at

Texas A&M University. I was not sure what to

expect, as every member of staff at the three

airports I landed in who saw my final

destination asked the same question, "why on

earth are you going there?!" My answer was

simple, because there was an amazing

entomologist based there. Dr Tomberlin has quickly become one of the most highly

respected forensic entomologists in the field and has a large laboratory of extraordinary

researchers. People from all around the world want to visit his FLIES facility, so I felt

overwhelmingly honoured that I was able to visit and get to known him and his research

team a little better.

I landed at about 5:30pm having taken four flights on the way. Dr Tomberlin kindly met

me at the airport and I checked into my hotel and went for some food. This gave us the

opportunity to have a chat about what I wanted to achieve from my visit and discuss

research activities I could due during my stay.

I was picked up very early the next morning

(Saturday 7th July) where we had a two-hour drive

to the Texas State Anthropology Centre.

This truly was an amazing experience for me as we

do not have these facilities in the UK and we are

only allowed to lay out animals (usually pigs) in

order to study the decomposition and insects they

attract. I was a little nervous on the way as to how I would react to such a sight. Throwing

up in front of a host was something I desperately wanted to avoid! I have only ever

encountered a corpse at an autopsy and this would be quite an unusual environment. As we

pulled up to the sight, and entered the double set of large metal security gates, there were

vultures


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circling the warm air above and a distinct sweet smell in the breeze. It was an unbearably

hot day and Jeff promised we would be out in the heat sampling from the bodies for less

than half an hour. He showed we around the sight, showing me the various stages of

decomposition the bodies were at. He chose the freshest body there to collect samples to

take back to his class. This was an amazing chance for me to see how to collect insect

samples from a human body, something I have never experienced before. I am pleased to

report I coped well with my first experience of an anthropology facility and it was

unbelievable to use such an amazing source which has aided scientific advances in many

areas such as decomposition, anthropology, FBI training and forensic entomology. Sunday

involved identifying the collected samples and going over my first case report with Dr

Tomberlin (he clearly has never heard the phrase, "Sunday is the day of rest...")

The following week involved some intense work,

including the collection of hydrocarbons (my PhD

research area) from six different species which I shall

analyse later in my trip at JEOL. I reviewed five case

reports and really enjoyed seeing the dynamics of the lab

in action. Dr Tomberlin's research group are the most

inspiring group of people I have ever come across. They

are extremely driven and ooze passion for their research.

The evenings consisted of socialising with the research group, including playing pool,

bowling (both of which involved Dr Tomberlin losing), cinema and some bad baking (yes,

that is a screwdriver in the picture below!)

The group will be sorely missed as I truly felt at home and so welcomed by every single

one of them.

Bad baking in Texas!

Dr Jeff Tomberlin

Firm cakes!


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North American Forensic Entomology Association

I arrived in Las Vegas on Monday

16th July to attend and present at the

North American Forensic

Entomology Society (NAFEA). It was

my first visit to Las Vegas and was

certainly a trip to remember!

Tuesday afternoon consisted of a

workshop on statistics, which was

hard work but very useful and

productive. The oral presentations took place on Wednesday and Thursday. There was a

wide range of research being presented and it was amazing to network with such

inspirational scientists. My oral presentation on Wednesday seemed to go smoothly

although, as always, I was a little nervous! I was therefore extremely shocked to have been

awarded the top student presentation for the Percival Molecular Forensic Entomology

prize! There was a kind donation of $300, which shall be saved so I can attend next years

NAFEA conference.

The Texas A&M FLIES group


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The mixer on the Wednesday evening was a great chance to meet with all the attendees

and the location of the Flamingo hotel (where the conference was held) was perfect for

many great nights out that were had during the week. I was also fortunate enough to

experience a helicopter ride over the Grand Canyon which was breath taking.

A memorable conference where I was fortunate enough to meet some leading researchers in

the ever expanding field of forensic entomology. I certainly hope to be a regular attendee of

this annual conference.

Socialising with some student at the drinks mixer

Presenting my talk at NAFEA


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Michigan State University

I arrived in East Lansing on 22nd July.

Having had no time to recover from Vegas,

I was picked up by forensic chemist, Dr

Ruth Smith in the morning of 23rd July. We

spent the next day and a half looking at

statistical techniques, in particular,

Principal Component Analysis. I also got

shown around the highly impressive Mass

Spectrometry unit at the University, which

is considerably better equipped than most institutes I have visited.

The afternoon of 24th July was spent with Dr Richard Merritt, who was the main reason I

wanted to visit MSU. He is one of the most experienced and renowned aquatic

entomologist, who is also very well known in the field of forensic entomology, and has

worked on many cases over the years.

Myself with Dr Richard Merritt


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I was also fortunate enough to spend a morning with Dr Foran, a forensic biologist at

MSU who has a very busy lab of enthusiastic masters students.

Over the course of the week, I was shown around the wonderfully large campus of MSU,

went over some case work with Dr Merritt (something I cannot do in the UK, as students

do not get access to casework), visited the MSU arab breeding farm, had great food with

Rich and his lovely wife and watched some baseball!

My time spent with Dr Merritt, Dr Smith and Dr Foran seemed to fly by all too quickly,

but I took as much as I could from each one of them as they are all very knowledgeable in

their own areas. I was honoured to spend time in their company.

Dayton University

My fourth visit (July 30th to August 2nd) was at Dayton University to spend some time

with assistant professor, Dr Eric Benbow and his research group. Dr Benbow has a broad

range of research interests ranging from aquatic ecosystems to ecological communities and

forensic entomology. His research team is very active and keen and an absolute pleasure to

be around, like many of the students I have met in the USA. I am always overwhelmed by

the passion they have for their research.

My time with Dr Benbow was spent going over some statistics as he and his post doctorate

associate, Dr Jen Pechal, excel in

this area. I was also able to do some

more hydrocarbon extractions of

species of blowfly commonly found

on decomposing remains in this

region. The team had a scavenging

experiment currently running whilst

I was there, and I was lucky enough

to go to the field one evening to see

the laid out pigs. The maggot


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masses were in full swing, which I found very interesting (big thanks to Jamie Alfieri for

allowing me to tag along!) I was also able to collect my own pupae from the ground around

the pigs...something I have never had chance to do in the UK, so again, this was a great

experience for me.

Time was also spent planning future experiments which will hopefully lead to a publication.

I certainly hope to collaborate more with this group as they have some amazing ideas and I

feel I could really learn a lot from them.

In the evenings, we went to a baseball game, played some pool and Dr Benbow enjoyed

some karaoke and dancing.

It was fantastic to spend time at Dayton University and once again I cannot speak highly

enough about this fantastic research group, who's work is seriously impressive and I look

forward to having a great working relationship with them in the future.

Collecting some pupae from the soil with the Dayton team

Eric Benbows research team


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Ohio State University Police Well, what can I say about this visit...it was short, but very, very sweet! I spent one day

with Captain David Rose of the Ohio State University Police. Former fellow, Dr Andrew

Hart, suggested I visited Captain Rose and I am very glad I took up his recommendation.

So much was packed into the day and it was a lot of fun to be driven around in a Dodge

undercover police car!

The day started off with a tour of the police station followed by a behind the scene tour of

the imposing Ohio State Football Stadium, home of the Buckeyes (capacity of around

102,000!!)


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We then headed over to the Bureau of Criminal Investigation Laboratory where Captain

Rose kindly arranged a tour. We saw

everything from the firearm and ballistics lab,

to the DNA lab, to the chemistry lab. It was a

seriously impressive set up and an absolutely

amazing experience for me to have a one-on-

one tour. Many thanks to the laboratory for

allowing me to visit.

The afternoon was spent having another behind the scene tour, this time at the Universities

basketball stadium, as well as the glorious main library on campus. The students are very

spoilt having such an amazing place to study. We also headed over to the anthropology lab

where Captain Rose spends a lot of his spare time. As well as working full time for the

police, he managed to study for his undergraduate and masters degree in anthropology and

has recently started his PhD studies. I find this extremely inspirational for someone who

works such long hours in a highly demanding and challenging job. Hats off to you Dave!

We paid a visit to the Chemistry building to see who was about and managed to have a

brief chat with some analytical research students as well as Professor Susan Olesik. To

wrap off a wonderful and productive day, I joined Dave and his lovely wife, Missy, for

dinner. I would like to thank Dave for hosting me for the day. It was a very enjoyable

experience for me.

Captain Dave Rose


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JEOL, Peabody, Massachusetts

JEOL is a leading global supplier of scientific

instruments used for research and development

in the fields of nanotechnology, life sciences,

optical communication, forensics, and biotechnology. I was fortunate enough to visit the

company for two weeks.

For the first four days, I attended a training course on Direct Analysis in Real Time

(DART), given by the co-inventor of the instrument, Dr Robert (Chip) Cody, and assisted

by Dr John Dane. The course was attended by three other people, so it was a very small

group, meaning a lot of hands on experience with the DART. This course covered a lot of

material in a short space of time but by the end of it, I knew the ins and outs of

AccuTOF DART (Time of Flight Mass Spectrometer), as well as being comfortable

running my own samples (empty pupal cases collected from Texas A&M University and

Dayton University), changing parameters to suit my sample requirements and analysing

the data obtained.

The remainder of my time at JEOL was spent having some invaluable one-on-one time

with Chip and John on the DART as well as a number of other instruments including one I

am very familiar with from my PhD research, Gas Chromatography - Mass Spectrometry

(GC-MS). I also collected some up close and personal images on the powerful Scanning

Electron Microscope (SEM) and also some Matrix Assisted Laser Desorption/Ionisation

(MALDI) data. The wealth of information I was able to gather from my samples was

unbelievable and to have so many experts at hand was truly mind blowing.

Myself, Chip, John and the three attendees of the DART workshop course


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SEM image showing the posterior spiracle (one of a pair) on the empty pupal case from the

blowfly species, Chrysomya rufifacies. The pupal

case is the hardened skin of the third instar

larvae. Larvae use these spiracles to breath when

they are submerged in food and are often

mistaken for eyes. The number of slits visible

indicates the instar (1 slit for 1st instar, 2 slits

for 2nd instar and 3 slits for 3rd instar). They

are also used a morphological trait to enable

species identification.

The results I was able to collect were exceptional but the best results came on the second

to last day in the lab (typical!) Chip and I are very excited about them and they will be

published in the near future.

I would like to thank JEOL for accommodating my visit, with special thanks to Chip and

John for helping me so much and giving me so much of their time. The results gathered are

mind blowing and I cannot wait to see them published! I hope this is the start of a very

exciting collaboration. Seeing the co-inventor of DART working away on his own

instrument is an experience I will never forget. He has an exceptional mind and he is

indeed the "God of DART"!

SEM image

Myself with Chip and John


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Final thoughts…

Before I left for America, and upon returning to the UK, I have appeared in a number of

newspaper articles, press releases via chemistry and forensic science societies and had a

radio interview. In doing this, I hope to spread the word of the WCMT and urge others to

apply. I hope to continue to promote the trust as best I can.

I have met some wonderful people on my travels and words fail me when I try to explain

my experiences. It has certainly exceeded all expectations and that’s all down to the

WCMT for giving me this life changing opportunity. I have made many fantastic

connections with some outstanding scientists and I really hope I can bring my knowledge

back to the UK to benefit and enhance scientific developments in my research field.

Winston Churchill was a man of perseverance, bravery and persistence. This inspirational

figure remains a role model to many, 47 years after his passing. My lack of science

background prior to university has led me to hold a lot of these traits and I really have

learnt that anything is possible if you apply yourself and want it bad enough.

Applying for the fellowship has, hands down, been the best thing I have ever done and I

have so many astonishing memories that I will never forget…Thank you

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