Flying High in Histology

December 9th 2014

Forensic specialist, Michael Pais, explains how a new histological method can help forensic scientists determine the age of fly eggs to more accurately predict the time of death.

 

Michael Pais

Michael Pais, Forensic Histology Scientist at VIFM.

Research Project:
Histological Method for Ageing the Eggs of Calliphora Vicina Robineau-Desvoidy (Diptera: Calliphoridae) Using Qualitative Determination of Anatomical Structures.

Forensic Specialist:
Michael Pais, Scientist

Department:
Histology, Forensic Services, VIFM
 
 
 

Abstract:

 
The ageing of blow fly eggs may be critical to a forensic investigation, but there are currently no forensically useful timelines describing anatomical changes in embryological development. This is mainly due to the lack of a cheap, rapid and technically simple technique to allow mass production of slides for research and casework. We present a histological method that uses a slightly modified standard laboratory run with 1-hour fixation in 10% formalin, 2-hour softening in Molliflex and H&E (hematoxylin and eosin) staining. We also present a summary of the anatomical changes that can be visualised in the developing eggs of Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) using our technique. We examined eggs from at least three different females grown at 15 degrees C and sampled at 6hr intervals, and eggs grown at 20 degrees C and sampled at 3-hour intervals. Histology tissue cassettes containing eggs were cut through in 4 µm  thick sections at levels 48  µm apart. We were confidently able to visualise unique anatomical features that defined each of the major stages within cleavage, blastula formation, gastrulation and organ differentiation. Artefacts encountered include shrinkage, shatter, tissue loss and knife lines. Tissue loss prevented ageing of embryos in only 7% of slides, and insufficient anatomy was seen in only 2% of slides.

 

Firstly, tell us a bit about yourself

I’ve worked at VIFM for 11 years now in the Histology department. I got into histo (as those in the industry like to call it) more by accident than by design. I wanted to do something related to medicine but not medicine itself. I found out about an RMIT course called Medical Laboratory Science and decided to give it a go. I really enjoyed the course but histology was the subject that I really developed an affinity for. I took it as a major and have never looked back.
 

Explain your research project for the non-sciencey people out there

So – sometimes after death, the body may be colonised by various insects. One of the first to colonise a body are blowflies. If the female fly lays eggs on the body, and they have not yet hatched, then the minimum time of death can be determined by aging the eggs. We can use this knowledge to estimate how long a person has been deceased. Using histology, which is a relatively simple and cheap method, we can analyse the eggs of the blowflies and more accurately predict time of death.

Histology looks at the cellular structure of tissue. We can use this to look inside the eggs and determine what stage of development the eggs have reached, and thus predict how long the eggs have been on the body. We can extrapolate this knowledge to then calculate a likely time of death.

 

How did you become involved in this particular project?

The histology lab wanted to get involved in doing some research and, at this stage, no concrete projects were available. I was talking to Dr Melanie Archer (a Forensic Entomologist) about this and she mentioned that she was interested in doing some research on blowfly pupae. Well, after much discussion and forward planning, we got the project up and running. The fly eggs were originally an adjunct to the main project but, for various reasons, we started with the eggs and our results were so promising that we decided to focus on the eggs rather than on the pupae. We will come back to the pupae at a later stage.

 

Calculating the age of fly larvae is nothing new in the field of forensic entomology, so what is so different about the technique you’ve used in this project?

You’re quite right there, working out fly larval age is not new. What is new is working out the age of fly eggs instead. Also histology has not been effectively used in determining fly egg age at particular temperatures before. This is very novel and most importantly, cheap and effective. This method would allow any histology lab to run many, many samples cost effectively.

 

Fly egg under the microscope

Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) aka a fly egg which has undergone the histological processes in order to highlight the various stages of development within the egg. Histology is a method of producing ultra-thin sections of stained tissue mounted on glass slides ready for microscopic examination. The tissue sections are stained with a variety of specialist dyes to highlight various cellular structures and make them easier to see under a microscope.


 

What were some of the biggest challenges you encountered throughout this project?

The first and biggest challenge was finding the time to get started. The project had to fit within the core work and this was made all the more difficult as we started this project in the middle of the building re-development. These two factors alone significantly slowed down our work-rate.

It was also difficult working out what structures we were seeing. We used old journals and textbooks (one from 1895) to attempt to visualise the structures. Of course it’s very different looking at idealised pictures and composite photos of things and then seeing them in real life. Many times Dr Archer and I sat there racking our brains, trying to work out what we were looking at.

 

What aspects of your findings surprised you the most?

Originally, we were going to look at blowfly pupae, and as an adjunct to this we thought we’d take a quick glance at the eggs. For various reasons we started with the eggs and to our surprise, we landed on a goldmine of information that was relatively untouched. When we researched the literature, we found very little relating to blowfly eggs and so it was decided that we would focus on the eggs and come back to the pupae at a later date. So far, we haven’t yet managed to get onto the pupae as the eggs have become our prime interest.

 

What do you think could be the practical implications for this new technique in the future?

This work will allow us to better determine the time of death of a person who has either been dead a short time (under 24 hours in warm weather), or up to a week in very cold climates. If a body is found, and at that time there are no maggots or pupa on it, the eggs that are likely to be there can be used to determine time of death.These results could then be used, for example, in legal proceedings as evidence of time of death. We can also use this procedure to determine time of death in old cases as long as the eggs were collected when the body was found.
 

You took this research paper to a conference recently. When and where was that and how did it go down?

I went to the Australian and New Zealand Forensic Scientific Society ( ANZFSS) International Symposium in South Australia in September this year. I gave a talk on this topic and with the help of Dr Archer.

The talk went very well but I was very nervous as this was the first time I’d spoken at a conference. I actually enjoyed the experience and even managed to pick up an award. That was very gratifying.

 

Where to from here? Will you continue to refine your methods in this area? Will you be involved in any more forensic entomology projects? Will you become “The Fly”?

[Laughs] I don’t think I’ll become “The Fly” any time soon but I will continue to pursue this project further.   Our next port of call is to test our findings by doing a blind study to see if we can actually predict the age of the eggs without any prior knowledge of how old they actually are. We will then shrink down our time lines from 3 and 6 hours between egg collection to 15 to 30 minutes between collection to get an even clearer understanding of when various cells and organs develop. Finally, we will do some special stains to look at other changes that may be hard or impossible to see with H&E staining.

 

Knowing what you know now, could you kill a fly?

Previous to doing this work, I did not hesitate. If a fly was anywhere near me….SPLAT! Now I realise that it is a living creature with a brain, muscles – all the things that make us alive. It feels pain, fear, etc. I haven’t killed a single fly since I started this project and probably never will again.

 

Photograph of Michael Pais and fly by Ashil Davawala, Scientist, Molecular Biology at VIFM.

 

LINKS

Read more about Forensic Histology