Advancements in Analysis of Human Remains
Learn about new approaches for more efficient analysis of skeletal remains in this webinar. Sheree Hughes from Sam Houston State University will present a comparison of different methods for DNA extraction from bone—with and without powdering, and with and without automation. Rachel Oefelein from DNA Labs International will share streamlined Rapid DNA protocols for analysis of human remains, enabling fast recovery of discriminating DNA profiles for time-sensitive investigations such as disaster victim identification (DVI). You will learn about: How different approaches for sample preparation and automation with bones can affect workflow efficiency and downstream STR recovery Streamlined Rapid DNA protocols, requiring minimal hands on time, to recover discriminating DNA profiles from human remains
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Hello and welcome to Advancement and Analysis of Human Remains presented by
Forensic and
Sponsored by Thermo Fisher Scientific.
This is the fifth webinar in the six-part Future Trends in Forensic DNA
Technology series.
My name is Michelle Taylor, the Editor-in-Chief of Forensic, and I will be your
moderator
throughout.
For today's webinar, you can earn one hour of continuing education credit.
Following the conclusion of the webinar, you will receive an email with
information on
how to obtain CE Credit Documentation.
We have a great lineup scheduled to present to you today, but before we begin,
I'd like
to take just a moment to cover a few logistics.
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Today, you will hear from Sherry Hughes, who is an Associate Professor of
Forensic Science
at Sam Houston State University.
Sherry completed a PhD in Health Sciences and Forensic Genetics at Bond
University on
the Gold Coast in Australia, investigating forensic DNA typing methods for
highly degraded
samples, such as those recovered from mass disasters, missing persons, and
historical
cases, in conjunction with DNA repair techniques and phenotypic S&P analysis.
In 2015, she was appointed by the Governor of Texas to the Texas Forensic
Science Commission.
You will also be hearing from Rachel Uphiline, Senior DNA Analysis and Director
of Research
and Innovation at DNA Labs International.
Rachel is currently a PhD candidate with Griffith University in Australia,
having received
her Master of Science in Forensic Science and BS in Criminal Justice with a
Forensic Science
minor.
Prior to DNA Labs International, Rachel worked at the Armed Forces DNA
Identification Laboratory.
Rachel is testified in 10 Florida counties, 3 states, and 4 countries as an
expert witness
for both the prosecution and the defense in misdemeanor and felony trials.
Thank you for attending the fifth session in the six-part Future Trends in
Forensic
DNA webinar series.
After the webinar, please be sure to check your email for more information on
CE Credit
documentation.
We look forward to seeing you on October 15th for the sixth and final part.
DNA Hit of the Year DNA Database Programs help solve more crime.
Without further ado, I'm going to hand it off to Sherry to get us started.
Thank you Michelle and thank you Therma Fisher for inviting me to speak today.
So I've been asked just to share some of the work that we've done in our labs
around
processing bone samples and how we've integrated Therma Fisher's traditional
STR workflow and
their DNA extraction chemistries with a little bit of a twist for multiple
applications in
our lab with some success.
So the majority of the work that I'm presenting today was performed by one of
my former PhD
students, Michelle, and my recently graduated Honors student, Madeline.
So the stock standard disclaimer, of course, and let's get straight into it.
So we all know that there are multiple challenges with skeletal samples.
Sample processing can be extremely long-lengthy, laborious, and can require
some specialized
equipment and training.
And often it will completely consume a precious sample.
We have a lot of PCR inhibition that we need to consider and with the
extraction, DNA extraction
method obviously needs to be effective of removing all of those inhibitory
agents.
The DNA we yield from a lot of skeletal samples is going to be in very low
amounts and can
be often be highly degraded or even damaged.
And this can lead to difficulties and challenges for downstream STR typing.
We also need to bear in mind the increased risk of contamination in these very
low-level
samples as well.
But also one of the steps in the sample processing of bone where we powder into
a very fine powder
can increase the risk of that contamination as well.
So of course the traditional STR workflow in the laboratory should look
familiar to you
all.
And the work I'm presenting is using all of these thermofisher kits and chem
istries on
these instruments.
All the way from extraction through to separation detection on the 3500.
So the general sample prep and sample prep of bone samples essentially always
starts with
the sanding.
So we would sand off usually the top millimeter or so of the bone to remove a
lot of those
external contaminants.
Then we would go through a series of chipping into smaller fragments, wash
series of bleach,
ethanol water and then crushing that bone into a very fine powder either with a
freezer
mill as you see here or a blender cup or with a drill.
So once we have that powder we then need to extract the DNA from that powder.
And one of the industry standard methods now is what we would call a complete
or total
demon or demonization protocol from bone powder which is pretty much used in
the majority
of bone labs around the world now.
In terms of it was originally described by Lorella Tl and has been further
refined by
that same group and described and used by many many other researchers.
And essentially it's relying on an increased concentration of EDTA to maximise
that decosification
and mineralisation and completely de-mineralise that powder.
Now the prep file of EDTA kit was released by Thermo Fisher and it was
developed with
a very aggressive lysis buffer as well for bone and tissue samples, bone and
teeth.
And again we wanted to incorporate this in our lab for various projects and
again both
of these methods are relying on extracting from bone powder.
However there are times where we would like not to have the powder.
So labs may not have the equipment to crush bone into fine powder.
So this is also really interesting method or approach for precious samples that
you may
not want to destroy.
So maybe some museum or ancient remains you don't want to be totally destroyed.
So maybe this is something that could be used to extract DNA, still leach DNA
out of those
samples without maintaining the integrity of the sample itself.
You can also, this may also reduce the potential for contamination if you're
not powdering and
creating all those aerosols of bone powder.
And also interestingly you can then go back and re-extract from that same
sample a second
time.
And so a few people have looked at different approaches for this with bone and
teeth as
you can see here.
And then a little while ago the T-bone X kit was released, was developed
specifically for
this reason, to be able to extract DNA from whole bone tissues without having
to powder.
And so it's an initial digestion step that you would then pair with another DNA
purification
step.
In our case we wanted to look at the combination of the T-bone kit with the Pr
EPFYLA BTA kit.
So the first project I want to talk about is exactly that.
It's looking at the results for STR typing with powder-versing chips, using
powder-versing
just chips, so being able to avoid that powdering step.
Look at BTA, PrEPFYLA BTA alone, or with the T-bone buffer, is there going to
be an
accumulative improvement of the T-bone kit?
And then look at manual versus an automated approach.
So we collected six bones from skeletonized remains out at Staphs at San
Houston State
Uni, and we subjected them to three various, those DNA extraction methods.
So the total demineralization protocol from powder, PrEPFYLA BTA from powder,
and then
PrEPFYLA BTA combined with the T-bone buffer from the chip.
And then combination of manual versus automated downstream purification on the
automated express.
So looking at our control method, so we would crush the bone powder and go
through your
demineralization protocol manually for around about 30 hours or so that would
take.
Then using the PrEPFYLA BTA kit, again from bone powder, threw down to sell l
ysis at the
BTA kit, but now looking at introducing the T-bone kit prior to PrEPFYLA, we of
course
are able to avoid the crushing and the processing of the tissue into a powder,
added them to
the demineralization and digestion with the T-bone kit, and then both of these
then are
subjected to the PrEPFYLA BTA kit, either manually or automated, is what we
were looking
at for our comparisons.
So as expected, we did see a significant decrease in the amount of DNA that we
recovered when
that process was automated.
And this wasn't unexpected.
Bone labs pretty much would traditionally use a manual extraction procedure for
this
very reason, for the fear of losing whatever little DNA we've got.
We essentially don't want to be losing due to robotics.
However, when we looked at the STR success from those DNA extracts, we found
that there
was no significant difference at all between the STR success based on those
methods.
So the PrEPFYLA BTA was the quickest overall.
Even though we had to powder, it was still the quickest, we could avoid the
overnight incubation.
But more importantly, the T-bone X kit really didn't have any additional
benefit over BTA
alone.
However, the BTA PrEPFYLA kit was still dependent on powdering the bone.
So we wanted to look at, well, if the BTA buffer is really good at digesting
from the
bone powder, is it aggressive enough and effective enough on bone chips without
having
to powder it?
And then if so, will this number and the size and the number of chips that we
use for digestion,
is that going to make a difference?
And if we increase the incubation of the initial incubation step because of the
chips versus
the powder, could that improve our results?
So we took a bone from a badly burnt body, sanded and processed it down, and
then we looked
at a different combination of different size chips and the quantity of chips
and then that
incubation time.
And coupled that with our PrEPFYLA BTA extraction.
So we had our bone powder and our bone chips, we mirrored the amount.
So we had 50 milligrams of bone powder and we also had 150 milligrams of bone
powder.
We also had 50 milligram bone chips, so one for the 50 milligram, three to make
up the
150 milligrams and then one large 150 milligram powder, arm chip.
And then we looked at increasing the incubation from the recommended two hour
protocol through
to a doubling of four through to an overnight incubation.
And what we found is that there was no significant difference in DNA yield or
STR success based
on any difference in the incubation time or the combination of chip number and
or size.
The only difference we did see as what we expected in the DNA concentration,
when we
got our DNA yield, we recovered less DNA from 50 milligrams than we did 150,
but there was
no difference at whether it was a powder or chip.
So none of the alternate variables that we looked at actually generated any
significantly
better results than the initial manufacturer's existing protocol of 50 millig
rams of bone
tissue with a two hour incubation.
But now we were able to demonstrate that we did not need to chip to powder that
50 milligram
bone we can we can extract from the chip itself.
So then we were asked, well what else can we do with this protocol?
Perhaps me, if they don't have the equipment to powder their bone, could they
use this
as a initial very quick rapid screening tool to decide, well, what are the
likelihood of
the of STR success from these bones that we may have?
Or really do we need to send them off to another lab to do some Mito or MPS or
some other type
of specialized preparation?
And so we wanted to look at can we use this bone chip on a very quick automated
process
on the automate?
And then it was, well, what other bone what other skeletal elements may this
work for?
Not just your traditional bony elements and your long bones, but any other
parts of the
body.
So we wanted to look at that as well across different environmental insults.
So again, we started off with our controls.
So we have our powdered controls, our 50 milligram samples, one that were
processed either
with our total demon method or our prep file or BTA method manually.
Then we wanted to look at, okay, our screening method is using a very quick 50
milligram
chip, no powdering straight onto the automated on to the automatic express
using the prep
file or express chemistry.
And then processed the same for our quant and STR typing, of course.
So what we found using this automated screening method with the chip is that we
didn't find
any significant difference in DNA yield.
As you can see, there's a trend for less from the chips compared to our control
methods.
But when we looked at the STR success, we did see a slight variation in our
results.
So as you can see, powder versus the bone chip, we did have more samples that
were generating
more complete STR profiles using the full bone powdering and manual method.
However, when we wanted to look at what's the effectiveness of a very quick
screening
method using a chip, we found that just under a half of the samples we tested,
we found
that we're generating more than 95% complete profiles from the chip alone on
the automated
express.
And if you end over just over half, so 13 out of the 20, we're generating more
than 75%
complete STR profiles.
So we thought that this method, being able to, the advantage of being able to
avoid the
powdering, was providing a very quick and easy screening tool essentially.
Then an interesting observation we had was looking at these traditional versus
non-traditional
skeletal elements for extraction.
So our traditional elements were your long bones, your compact bones, the shaft
of your
long bones that are weight bearing, so traditionally your femur and your tibia,
but also your
humerus.
But we also wanted to look at your non-traditional bones, so your hand and feet
for landage,
your fingers and toes, your carpal-submitted tasas, broken tooth fragments,
your vertebra,
ends, bits, other bones within the body that wouldn't traditionally be your
first choice.
And what we found is that non-traditional elements appeared to be yielding
slightly more DNA
and slightly less degraded DNA, as you can see from the slightly lower DI and
quant values.
However, these were not statistically significant in any way, but what was
significant was an
increase in our STR profile completeness.
So the number of reportable alleles, we actually got more complete profiles
from these non-traditional
elements.
So this was great because it means we can survey from much wider selection of
skeletal
remains when we have them in a case.
And this has been, this is not a surprise, we have, this has been shown by
multiple researchers
and reports that other non-traditional skeletal elements can be used very
successfully for
DNA typing.
This is more information on everything I've presented thus far has been, we've
also published,
so feel free to read that if you want after more information.
But then we wanted to look at something slightly different.
So we were asked to look at using our large range of environmentally challenged
skeletal
remains at using with global filer at the time, that it was unknown on the,
what the effect
of increasing the cycle number from the recommended 29 cycles of global filer
with some compromised
bone samples, what was the effect?
So we got asked to look at that and we looked at several increases because of
course it's
increasing our PCR cycle number, is your most common technique used for trying
to increase
your allele recovery from low template remains.
So we surveyed, we took about 12 bone and tooth samples from cadavers that had
been subjected
to a lot of different environmental insults such as burial, burning, embalming,
decomp,
etc.
We processed these in our stock standard method, we powdered these bones and
then we performed
extraction in duplicate.
So what we found not surprisingly is that overall as peak heights did increase
as the
cycle numbers increased of course and also the number of reportable alleles
increased
with additional cycles.
But this obviously was, the greatest increase was observed under 120 or so pico
grams which
we would expect.
And on the flip side though, as again as expected, was that we saw an also
increase in the number
of artifacts that we saw as we increased our cycle number and the amount of DNA
amplified
increased.
So we were able to sort of confirm that trend and have a look at the variation
from 29 to
32 cycles, we would see an increase, we saw an increase in stutter as we of
exaggerated
stutter and we also saw an increase in drop in as we increase the cycle number,
pull up
of course and we did see off ladder events as well increasing the more we would
increase
our cycles.
So here's an example of a say 60 pico gram sample with 29 cycles recovering
around about
just under 35% of their alleles with no artifacts increased to 30 cycles we
were able then to
recover another 20% also without increasing seeing any more unwanted artifacts.
Up to 31 cycles we were then able to recover 70, just just over 70% of our
profile but
we now start to see artifacts and then of course increasing further increases
those artifacts
but we're not seeing any increase or much improvement at all now number of alle
les recovered.
So this work helped them officially then tweak some of their protocols and also
an application
note on these and we also published these results as well.
So of course our HID tools could be used for multiple applications, everything
from our
routine forensic casework and crime scene investigations through to missing
persons cases and mass
disasters solving historical cases and something a little bit different is
using HID for
improving the traceability and tracking of specimens within an anatomical
collection.
So usually what happens in a facility body donor program is when they are
received the
bodies are assigned a unique identifier and tagged upon arrival.
Then those bodies go through the embalming process and then when they are then
housed
and used for research and teaching those bodies are then dissected and will be
separated
into various areas and containers.
So of course those tags then are the isolated organs are tagged and this
identifying number
obviously is then associated with that original donor within within a registry
but does it
go wrong?
Well every now and then it has and of course this is an extremely serious issue
we want
to make sure that bodies and body parts are correctly assigned to their donor
so that
can be cremated and returned obviously to families as required and of course
when this
has happened in the past very isolated incidences DNA typing has been relied
upon to then try
and re-associate those remains to the non donor.
But as far as I'm aware there is no anatomical or body donor program in the
world yet that
routinely has established and uses a DNA database and tracking system to
establish the identification
of all of their donors and their specimens within the collection.
So this of course could be a way of improving the traceability of these organs
within a
facility and increase and really boost their quality assurance programs.
So how this project came about was the university I was recently at has an
anatomical program
within what's called the gross anatomy facility and essentially wanted to know
could we establish
this type of program here.
But we didn't know what the spread of the DNA quantity and quality and recover
ability is
going to be out of the remains we had within the facility.
So this was essentially a survey to see what we had and what we could do.
So the gap within the biomedical sciences at the University of Queensland is
the largest
anatomical, persected and skeletal collection within the state of Queensland
and we recover
or receive approximately 100 bodies per year donated to the program and these
bodies can
be and samples can be maintained for up to 10 or 20 years before being returned
or disposed
of.
And so these specimens can be anything from whole bodies through to persected
limbs and
torso etc. various body parts we have isolated organs that can be preserved
within pots and
of course skeletal remains and collections.
But these specimens are all embalmed and protected and preserved with embalming
solutions
which could either be commercial or in-house versions.
So there's a lot of variation.
But embalming of course is there to preserve the human tissues.
It's there it's used by within funeral homes within these types of anatomical
collections
and pathology to preserve the tissues but it also greatly degrades and damages
the DNA.
So how these embalming works usually falls within two general methods.
So the whole body or the organ is embalmed in the, is immersed in the embalming
solution
or the solution is flushed to the circulatory system for diffusion.
And a most common agent used is a formal and based preservative and of course
as we know
that creates extreme DNA damage and degradation particularly with cross-linkage
and fragmentation.
And as most of us would be aware that Aftil have also encountered this problem
with a
lot of their remains that they've recovered their skeletons and bones from the
punch bowl
where these bones we know have been immersed in these really really aggressive
formal and
based embalming solutions and powders.
So this project really started by a survey of what was in our gaff and what is
the likelihood
of being able to recover any sort of amplifiable DNA.
We sampled hundreds of tissues across the body but because this is a bone talk
I wanted
to focus more on those.
So out of all of those we had 72 bone samples that were recovered and these
range from
bodies that were embalmed for up to, from anywhere from one week through to
about 13
years.
And we employed the same DNA extraction method as what I previously described
in this talk.
So using the bone chipping method with the BTA kit for bone extraction, for DNA
extraction.
We recovered a range of DNA concentrations as we would expect but the majority
of the
samples we actually recovered more than 100 pC per microliter to be able to
amplify for
STR analysis.
We had very few samples with very small amounts of DNA.
However the DNA degradation index or the DIY was rained quite substantially
from anything
from very mild to moderate degradation to extremely high degradation and some
of these
samples were so degraded we couldn't even get a DIY value for.
However we did not detect any PCR inhibition at all so we were very confident
that the
extraction process was very efficient.
So in terms of the results from the bones and cells as you can see around about
three
quarters of the samples we recovered more than 90% of the cells in our STR
profiles and
only a few of the samples we generated less than 30% of the cell recovery.
And these samples were the samples that were much older have been embalmed for
much longer
between four and 13 years.
But 65 out of those samples were able to be positively identified back to the
donor.
So out of interest though, all the tissues as a whole what we did find was that
when
we looked at the quality of our STR profiles these did generally decrease over
time but
it was actually more dependent on the tissue use whether it was skin or muscle
or lung
or liver which obviously I don't have time to talk about but it was extremely
interesting.
Another little interesting thing we found was that we recovered a lot more
higher quality
DNA that led to more complete profiles when we recovered the tissues from the
lower limb
versus the torso or upper limb part of the body.
So we thought that was interesting that needed to be that warrants further
investigation.
So really to summarise this talk we found that the PrEPFOLA BTA kit has been
extremely
effective on a very wide range of skeletal material that have been exposed to
lots of
challenging environmental insults and we were able to do this with in without
bone
powdering.
So we found that the BTA kit is extremely aggressive in terms of its lysis and
effective
and recovering DNA from these bones without the need to powder.
We've also demonstrated that increasing the PCR cycle for these challenging
bones does
improve the recoverability of alleles without introducing too many more
artifacts.
We also found that the PrEPFOLA BTA forensic kit was very effective in being
able to use
for recovering DNA from these embalmed samples within an anatomy facility for
samples that
were embalmed for up to a year.
We generated a very successful STR results.
After that the samples we would assume would need a little bit more work to be
able to
get a definitive identification, most likely down the route of SNP typing and
use buyer
and PS but it does show potential for being able to create a DNA registry to
establish
provenance of these types of samples within body donor programs.
So with that I'd like to acknowledge and thank them official of course and in
particular
Angie Sherry and Lucy for their support and over the time of the years and then
also the
Department of Forensic Science and staffs at Sam Houston State and all of
course the donors
and their families that made this work possible.
So with that I'm happy to take some questions but other than that I will hand
over to Rachel
who will be telling you some really interesting information about using rapid
DNA technology
for bone samples.
Thank you.
Hello thank you for joining us today and thank you for that introduction.
My name is Rachel Afeine and I'm the Director of Research and Innovation at DNA
Labs International
and I'm going to be talking today about the Rapid Hit ID System and our
evaluation for
processing of skeletal remains.
DNA Labs International is located in Deerfield Beach in sunny South Florida and
we're celebrating
over 15 years now partnering with law enforcement across the United States as
well as Caribbean
nations.
Our initial testing and evaluation began in November 2018 and then in May of
2019 we
actually had the Rapid Hit ID System installed.
We initially had a different instrument so the actual Rapid Hit ID System was
installed
in May of 2019.
We're currently finalizing our validation.
We had added on a couple additional procedures so that involved some of our
delays in getting
signed off but we're so excited now to be offering the Rapid Hit ID System in
our
suite of offerings at DNA Labs International.
So what is Rapid DNA?
The FBI defines Rapid DNA as the fully automated hands-free process of
developing a DNA profile
from a reference sample buckled swab without human intervention.
We develop a DNA profile under two hours for a body fluid sample as an
investigative aid.
Both bone extractions can take anywhere from 12 to 72 hours.
I have noted here that this is laboratory dependent but it's not actually just
laboratory dependent
because it's also bone dependent.
So when we get really challenging bone samples sometimes for example with our
demineralization
method after that initial incubation you might not have full demineralization.
So we can respite that sample and give an additional incubation period.
In addition to that a lot of bones yield very small amounts of DNA.
So one extraction might not even be enough.
You might have to do two, three, four extraction.
We have one genealogy case in particular where we had to do eight extractions
to get enough
DNA to go forward with genealogy.
Bone extractions are incredibly labor intensive and they require special
equipment and training
for handling them.
It also requires a designated screening area.
You would want to be screening bones right next to where you have high quality
samples.
At our laboratory our high quality low quality bones are separated as well so
that they're
not being done in the same area.
That goes for all the equipment that's used for screening them too.
So what technique are we actually using?
I've included a few pictures here.
This is one of my favorite things to try to explain to examiners and say I want
a cross
section of bone.
They're like what the heck does that mean?
I always try to say I think a lot of friends and scientists do this where we
compare everything
to food.
But I always try to say we want it to look like a short piece of a celery.
So you want just that cross section where you have the anterior exposed as well
We do a lot of times get full sets of ramegans on occasion or full femur bones
but really
it saves everyone a lot of time, effort and money to just submit a cross
section.
So I would highly recommend if your law enforcement or medical examiner system
has the ability
to cut that cross section for you to request it to be submitted in that way.
As we usually take about a two to three inch section and if any tissue or
debris is present
on the bone a simple initial cleaning is required so you can either scrape that
bone or again
use a cutting wheel to try to get that removed.
However if the tissue is pink and healthy looking, abort mission.
Go forward with the bone extraction.
Healthy pink tissue is much easier to extract than a bone so I would recommend
at that point
just removing the tissue and taking that forward.
The bone must be sanded using a gremmel tool to remove potential contaminants.
A lot of times we get remains submitted that were discovered by civilians.
It's maybe gone through the hands of multiple medical examiners, sometimes with
cold cases
from the 60s and 70s.
People had handled it with their bare hands.
So we want to get any of that potential extraneous DNA off the surface of the
bone.
In addition to that we also want to get anything off the surface of the bone
that could be
inhibiting our reactions.
So for example chemicals that might have been leached into the bone from the
soils or anything
that it could have been exposed to over time.
Sometimes we get remains that were prepared for embalming so we're going to
want to get
any of those materials off.
Really by the end of it you want it to look like a perfect piece of porcelain
china is
how I always describe it.
At that point you're going to have to go through multiple washes.
So we'll do water and ethanol washes and you're going to want that to fully dry
So if you are going forward for de-mineralization extraction we're going to do
a mixer mill
powdering method.
And if you have any moisture remaining really you're just going to end up with
a paste.
So we want that bone completely dry before it goes forward.
So the bone fragments are broken up and we do that in water and pastel and out
of smaller
pieces are pulverized into powder.
Powdering the bone exposes a greater surface area for the extraction reagents
to reach
resulting in a higher extraction yield.
Cleaning materials so you want to make sure all the materials and the hood that
we're
using is completely free of any remaining bone powder from previous samples.
Bone powder gets into everything.
It's very easy to have bone powder left behind so you want to make sure that
you're doing
multiple cleaning stops in order to ensure that you don't have cross
contamination between
samples.
So the types of things we're using for cleaning are bleach, soap detergents,
ethanol, cleaning
reagents as well as UV lighting.
And this process is performed inside a specialized hood with filtration
capabilities.
So any bone powder that's going into the air is going to get through that filt
ration
system so that we're not having a deposit of bone powder across the hood.
So going into a little bit of our validation study, what we looked at initially
for our
sensitivity is decasted.
I say it's decasted too because really with the rapid system, we treated it
like a new
PCR kit.
So we aren't actually analyzing on the actual instrument.
We're using gene marker on computers so we take the data from the rapid and
then analyze
that separately off instrument.
We did that for a couple of reasons.
One, we are not an endist participating laboratory.
So although we work with a lot of endist participating laboratories, we do not
have direct upload
to codus.
So really that feature doesn't serve a function for us.
And in addition to that, we wanted the ability to be able to edit our samples
and have a
higher pass rate.
So by analyzing off instrument, we're able to account for dropout peaks that
are below
stochastic thresholds and be able to make better use of that data.
So really we treated like a brand new kit.
We have our own analytical thresholds on our own stutter filters, our own stoch
astic
threshold.
And so moving forward into what we did with the bones is we have to add a high
quality
bone sample from a known source.
We ground the bone sample into a small chunk consistency.
For those of you that are wondering what the heck is a small chunk consistency,
don't
worry.
I've got a slide for you coming up that I'm going to explain.
And we've prepared two sets of bone samples weighing approximately 10, 50, and
100 milligrams.
I say approximate and you can see that they're pretty close, but when you're
dealing with
bone chunks, it's virtually impossible to get it to weigh exactly the same.
So there's slight variation there, but they're pretty close.
And we ran those samples through rapid using Intel cartridges, not the ACE
cartridges.
So the ACE cartridges, we're using pretty much specifically for buckleswax.
You can also do body fluid samples for those, but really the ACE is mostly
going to be for
buckles.
The Intel has a higher cycle number and a slight variation in the buffer for it
, so you're
getting higher interviews from that.
So when you're dealing with bone samples, which can be tricky, obviously we
want the most
sensitive option.
So we process the raw data and we looked at average heterozygous peak heights
as well as
sister allele balances.
And as I mentioned, we analyze a G marker off instrument.
We did actually look at it on instrument as well too and compared how our two
methods
differed.
So remember, I mentioned that small chunk consistency.
What does it mean?
The consistency is too fine and will clog the system.
So I always say think of a sea salt grinder like that fancy pink Himalayan salt
That's the consistency that you want for your bones to make sure that you're
not going to
clog the system.
So for regular full-demoneturalization protocols, we're going to run our
samples through the
mixer mill and get that super fine powder that is not how we want it for the
rapid system.
So how is the data?
Remember we have high quality bones resulting in full DNA profiles from all
amounts tested.
So again, that was 10 milligrams, 50 milligrams and 100 milligrams.
By high quality, I'd say this is probably the most common question I got from
people
when I talk about our rapid skeletal remains protocol.
I would classify high quality as a subjector, but really what we're looking at
is samples
from remains that were found within 12 months of the time of the individual
becoming deceased.
And we want it to be in good environmental conditions.
So preferably in a cool environment in the world, but if they are outside, we
want nothing
with high heat, fires, bogs, acidic soil, things like that that are going to
degrade
those remains.
And I would also add in the caveat of no chemical treatment.
So we have remains that have been treated with acid and oil drums, things like
that, that
is not going to be a good sample for the rapid hit ID system.
Here we have the first bone from 10 milligrams from Set A. As you can see, we
have a beautiful
profile here.
It was about 9.9 milligrams, just a really, really small amount of bone.
But I can see here the average pea kites for blue, green, yellow, red, purple
and overall.
Here we have Set B. So this is a similar size sample, but from the second set.
And you can see we have very comparable results and again, a full beautiful
profile.
Go ahead to 50 milligrams, Set A. You can see here we have a slight increase
now in the
pea kites and similar gun for Set B. Even though this was 47.3 milligrams of
slightly
less input, but you're really not seeing that big of a difference.
When we go to 100 milligrams, that's where things get a little interesting.
So you can see all those E's and X's, E's or X's are things where we've had to
delete
peeks and you can see there's a little bit of noise on that baseline.
So I'm going to give you a better zoomed in peak or picture as well of that,
but it's
increased artifactual peeks and edits.
So basically we were seeing a really high amount of artifacts when we got up to
100
milligrams.
So at our laboratory, we recommend sticking in that 10 to 50 range for high
quality remains.
Here's a zoom in on the yellow dye channel.
So you can see all these extra little peeks here.
Some of them aren't sizing.
So you can see there's a lot of OBS, but some of these are actually falling
into bins.
So you would need to be cautious that this wouldn't register as a mixture.
So again, this is another advantage of analyzing off the instrument is that you
have additional
options.
Sorry, excuse me.
A lot of allergies down here in Florida.
We have endless summer.
So here we have bone one at 100 milligrams and this is for set A. You can see
the red
dye channel too.
We have this split peak here.
So set B for 100 milligrams was pretty similar.
We have those increased artifactual peeks and edits.
Here zoomed in.
You can see that as well.
So the good news about this is we can identify these and make record of them.
So if you do have these artifacts playing up in your profiles, we have the
ability to
edit these.
But again, the idea is to just reduce your amount of input and then not have to
deal
with these artifacts at all.
And here we are on the red dye channel.
So you can see it's not limited to a single dye channel across the border
having quite
a bit of edits and artifacts.
I will give the disclaimer from those two that this was before we had set our
AT.
So our analytical threshold is actually a little bit higher.
So a lot of those might not have come in.
But for the really tall peeks where we're seeing that bifurcation, those are
absolutely
still going to fall in at that 100 milligrams.
Here we have our average peek heights plotted against the bone amount.
So you can see as expected, the higher the input of your bone amount, the
higher of your
RFUs.
And you can see pretty comparable between the two.
Set two had maybe a little bit less.
There's more around like 2,500 RFUs where set one was around 3,000.
And that one is almost 3 milligrams left.
But I think it's like actually just coincidence because they're pretty
comparable.
So I don't think those 3 milligrams really made or break it for those RFUs.
Another way to look at the data is this average peek height by locus versus
bone input.
Just going through.
You can see the 100 milligrams versus the 47.3 and the 93.
So overall peek height ratio versus bone amounts.
You can see actually across the board, regardless of our input, we've really
great peek height
ratios.
This is what we would expect to see from any well-performing kit.
So we're ranging from about 76% to 83%, which as you know, anything over 70% is
great.
So this is what we want to see.
Here we have dye channel peek height ratio versus bone input.
Again, really well balanced regardless of input.
We're having great peek height ratios regardless of dye channel as well.
And here we have the red and purple.
So how does this compare to our current methods?
So we have our original demineralization bone extraction method, which is an
overnight incubation.
The hands on time.
So taking out the bone sauce, taking out the cutting wheels, a dremel for sand
ing off those
bones and getting that clean surface, all those washes.
It's about three hours of hands on time.
I'd say that's conservative.
If you're brand new, it might be more like more four or five hours.
But additional processing time to obtain that fine powder.
So we're adding that mixer mill step now at this point.
And the other thing that's important to mention here is that we have multiple
tubes required.
So we're doing multiple phenol washes.
And because you have multiple tube transfers, you're exposing yourself for the
potential
for a contamination event.
So we want to limit our tube transfers as much as possible.
And we also brought online a large volume extraction method.
That involves a butanol wash as well.
And instead of being performed in microcons, we're now in ultra fours.
And this whole process was actually brought online specifically for genealogy.
We were finding for our investigative genealogy cases that sometimes we were
having to do
two, three, maybe even four extractions to get enough DNA to go forward with
genealogy.
So I don't know if you could see in the tiny camera, but DNA for a codis SDR
profile, you
need this much, whereas genealogy, you need like this much.
It's a lot more DNA.
So we wanted to bring on a method where we could get higher return DNA from our
extraction
yields and hopefully limit having to do multiple extractions.
So here's our current bone extraction method against the large volume
extraction method.
And you can see pretty much across the board here, now you can pretty much, it
's every single
sample we added increased yield.
And what's interesting here is that these are actually really similar inputs.
So for example, if you look at the first one, we have 170 milligrams versus 160
So we've actually put in a little bit less input for bone powder here.
And yet we're having a higher yield return.
So this is ideal for our genealogy cases.
Our original bone method we actually only use typically for teeth at this point
So here we have our current bone extraction method versus the large volume
protocol and
the rapid hut.
So this is looking at our inputs versus the amount of nanograms of DNA that we
obtain.
So you can see that rapid hut actually holds up pretty well when it comes to
the high quality
samples.
So what about quality?
None of this means anything if we don't have high quality, right?
So we have all samples, the correct profile was obtained.
We had multiple replicates of the same bone source producing consistent
profiles and we
had no signs of contamination.
So overall, a quality win when it comes to the high quality remains, we're
getting high
quality profiles.
We want to know though what happens to that bone after the test?
So the bone can be removed from the cartridge after testing.
If you haven't actually seen the cartridge yet, it's about this big and it has
an opening
where the sample actually goes.
So those bone chunks are not being demineralized.
We're not turning to a liquefaction of the sample.
We still have those bone chunks there afterwards.
We wanted to know if you could retest them.
So if you had a limited bone sample, for example, and you put that in and say
your run failed,
would you be able to put that into a new cartridge and then rerun the sample?
And we found that actually you can.
So this is multiple tests of the same bone sample.
I will say that we actually did third and fourth rounds of this too.
I don't think I have the date on this presentation.
You can see that your overall RFU's decrease with each test.
However, we're still getting a full profile.
So as long as you get a full profile, it's a win at the end of the day.
We're not typically going to be rerunning these samples all the time anyways,
but it's
nice to know that we have that option in the event that we needed to reanalyze
that sample.
It also means that you could technically retain that sample for all time and
keep it so you're
not having that deleterious effect.
Multiple tests saying the same bone sample through the rapid hit ISC system
using the
Intel cartridges, so here we just have those average peak heights per locus.
And this is a 10 milligram high quality bone sample just to put things into
perspective.
You'll notice at Y and L and DYS 391, we have zero RFU's.
If you're wondering why there's those dips in the piece is because this is a
female,
so we don't expect any RFU's at the YSTR locations.
So what other options could we look at for bones?
We wanted to see.
We spent a lot of time actually trying to see if we could get samples that were
low quality
or what we would say is medium quality bone sample, so maybe higher in age,
like 10 to
15 years, but was in really good quality conditions to see if we could get
those to work.
So we tried just running the bone extract through to see what kind of DNA
profiles we
would get from that.
We also looked at partially digesting.
So doing a round of demineralization buffer and an incubation of those bone
chunks and
then loading it into the rapid cartridge to see the advantage there would be
would skip
all the phenol washes, phenols and nasty chemicals.
So as much as we can limit that in our laboratory would be great.
So we wanted to try that out.
Bone extract is actually fine.
The partially digested buffer is fine for high quality.
However, for those medium and low quality samples, we're still not obtaining a
DNA profile.
So as mentioned, we looked at those low quality bones, we evaluated bone chunks
versus partially
digested and bone extract.
And although you can get the profiles from the bone extract at that point,
there's not
that much of a point into going that route.
All it does is save you a little bit of time off of loading on the actual CE
instrument.
So I would not recommend this for low quality or medium quality bones.
I would stick to your original methods where you're really going to save time
here and
get good results as those high quality samples.
So I want to throw in a mention here for the application note that Thirbo has
released
for bone sample processing and the rapid hit ID system with rapid intel
cartridges.
DNA Labs International in addition to a couple of their laboratories as well
participated
in sharing their research and their methods for how they process bones using
the rapid
hit ID system.
So I would say if your laboratory is evaluating whether or not to bring this
online, this
would be a great resource to see the different options that other laboratories
are using.
So what else can we do for use this for rapid testing and bones?
You might be wondering who cares if you save a day or two processing, what
would require
you wanting to have these quick results.
So re-associations a big one for us.
So small plane crashes, dismemberment cases where we're trying to re-associate
all the
fragments of remains back to one individual.
We're able to rapidly do that.
If you think of cases especially with highly fragmented remains, we might have
an individual
who has in 50 different evidence submissions.
This would be a great option.
A lot less hands on time and makes your people doing manual extractions
probably really happy
with you.
It's also great in disaster human identification where you're trying to quickly
get answers
for the families that have lost loved ones and then rush cases.
So we still have Buckleswax processed in our traditional methods in our
laboratory.
But if you have an individual that is a case where they need very quick results
, this is
a great option where you can get your full DNA profile in 90 minutes.
So what do we do then?
If it's disaster human identification, we will do parent child siblings, half
sibling
relationships so that we can compare to that unknown profile and identify
through kinship.
What else can you do?
So if you don't want to just only do bones on your rapid-hit ID system, it's
also great
for muscles, organs, I would say, tread carefully with a spleen or brain sample
because those
will clog the system as well.
So really with the heart and liver, what we were doing is just kind of mincing
the sample
and taking that forward.
But I would say samples like spleen or brain that kind of, for lack of a better
term,
when they go into the cartridge, I would recommend swabbing the tissue or the
organ opposed to
actually putting it into the cartridge.
These work excellent, we love teeth.
And then as I mentioned, those rush cases for buckles, swabs, or body fluids.
So what else is new?
I mentioned a little bit on our large scale extraction methods.
These are for very degraded bones and it's a large scale format to produce
higher yields
of DNA.
So larger quantities of DNA is what we need for genealogy testing.
It's also great when you want samples to go more than one direction.
So sometimes we want that SNP profile for genealogy, but we also need an STR
profile
to be submitted for codis upload potentially with a vendor or with the vendor
laboratory
going to the end is participating laboratory.
And we'd like to save extract if we can for future testing or defense testing.
So this is a great option where you're going to high yields a DNA to meet all
the needs
of testing for your case.
I just want to touch on our hashtag DNA opt in campaign.
DNA Labs International has launched this campaign in the past year to try to
encourage people
to opt in their profiles for law enforcement on JEDMATCH and family tree DNA.
So anyone and everyone can help solve crime.
You may even know someone who has been the victim of a violent crime or had a
loved one
who has gone missing.
These photos are the faces of the many victims of unsolved violent homicides.
It was our mission to find justice for them.
Opting in your DNA and JEDMATCH may help investigators to find a killer, a
rapist or identify the
thousands of unidentified remains and works around the country.
It really is truly just so sad, especially if you look at the NICMEC map and
see how many
unidentified remains of children in particular, but it dolts as well.
It's in the thousands.
So as friends and scientists, it'd be great if we can spread the word that opt
in for
law enforcement can help give answers to some of these cool cases.
Here are some references for you.
So I mentioned that application note released by Thermo.
There's a link to this here.
Also name of the DUD confronting the rallies of rapid identification of
degraded skeletal
remains by SUNY-ADSA.
And then a couple other resources just that I've mentioned here as well for you
to refer
to.
I'm happy to always answer any questions.
So as well, if you want, just give me an email or feel free to call as well.
And we're always happy to share information.
I want to give a special thanks to Forensic Magazine, Thermo Fisher as well,
for providing
us this opportunity to share this information.
And my team at DNA Labs are national.
Without you all, none of this would be possible.
So thank you so much.
And thank you for the entire audience for your attention today.
Thank you, Sherri and Rachel, for that great information and insight.
Audience, it is almost time for the Q&A portion of our webinar.
If you have not already, please take a minute to ask our presenters a question
using the
Q&A dialog box on your screen.
Unfortunately, Sherri won't be able to join us for the Q&A due to unforeseen
circumstances,
but we still got Rachel, so don't be shy with the questions for her.
Additionally, if you do have a question for Sherri, please do send it
regardless, and
we will make sure she gets it and provides an answer for you.
Why do you think about your questions?
For our presenters, we're going to throw up three polling questions for you
very quickly.
Our first question is, how many bone samples does your laboratory process per
year?
None, one to 50, 51 to 100, 101 to 500, or over 500.
Take a minute to go ahead and let us know about your laboratory.
I'll gather these results right here.
It looks like most of you guys are doing one to 50.
All right, let's move on to our next one.
Audience, how prepared is your agency to handle disaster victim ID situations?
Your options are not prepared at all, somewhat prepared, but need improvement.
Fully prepared, ready for anything.
Go ahead and take a minute to tell us how your agency handles the DVI
situations.
All right, let's see what you said.
That's great.
It looks like most of you, our majority of you, are prepared by needing a
little improvement.
I'll tell some of you, a good portion of you are fully prepared and ready for
anything.
Always good in today's world.
All right, last question before we get Rachel up here.
Our last question is, would you like to receive more information on today's web
inar topics?
Yes, please contact me for a demo on the rapid hit ID system.
Yes, please send me pricing info and technical specs on PrEPSILR BTA and Autom
ate Express
Forensic DNA Extraction System.
Yes, please send me pricing info and technical specs on the rapid hit ID system
Yes, I would like to discuss further with a live person.
We're known not at this time.
Go ahead and take a minute and then we will gather these poll results and let
you guys
see what you said.
Thanks so much.
That's great insight from you guys.
We appreciate you taking the time to do these polling questions.
Now we are going to get to the extra fun part of the Q&A section.
Rachel, we have a bunch of questions for you coming in.
My first question for you is, what is the oldest bone you have obtained a
profile from?
I say the oldest bone that I have obtained a DNA profile from was from the 1800
So really, it depends on the bone condition because then we've also had bones
that were
from the last decade where you struggle to get a profile sometimes.
So it really has to do with the environment that the bone has been in and how
the evidence
has been preserved.
Wow, that's incredibly cool.
It's fantastic technology on your part.
That's great.
Okay, so do you have any more tips for our lab researchers for evaluating bone
quality
prior to processing if age is unknown?
For us, it's really about the visual evaluation.
So a lot of times we have bones coming in.
We will ask for them to send pictures of the remains so that we can try to
select from
the photos so that we're not receiving full skeletons at the laboratory all the
time.
Our evidence manager would kill me if we did that.
So usually we ask for photographs, but then a lot of times you really have to
physically
see it in person.
So we're looking for discoloration, how thick and how dense the bone is.
If there's excess spongy bone, those are things that are red flags for quality.
When we're looking at teeth, we're trying to avoid any teeth that have cavities
in them
as well.
So those can all be signs that you might have a little bit of difficulty with
the bone.
Okay, gotcha.
So how do you power the samples without spreading the DNA particles throughout
the entire laboratory?
So this is a question that came in, I believe, for Shari.
I would feel free to recommend following up with her as well.
But we also use Dremels in our laboratory, so I felt like I could probably help
out with
that a little bit.
We use specialized hoods with filtration devices that our ventilators have
airflow.
So you can literally, as your sandings see the bone powder lifting up for
anything that
comes loose, but the rest that we have down in our working areas, collected
into wayboats
when we're doing that.
Okay, gotcha.
Well, while we're talking about sanding, once a bone sample is cleaned via sand
ing or
another method, do you believe it is necessary to use bleach rather than a more
gentle detergent
wash before patterning or getting the DNA extraction process?
We don't use bleach directly on the bones at our laboratory, so we do bleach
all the
tools that we use and the surfaces that within that hood has to be cleaned very
, very carefully
in between samples.
But on the actual bone itself, we use ethanol and water washes.
I'm always cautious of ever applying bleach to bones because I just worry about
it affecting
the quality of the sample.
I know a lot of laboratories do, but ours after sanding, I don't see any need
of doing any
washes outside of ethanol and water.
Okay.
Then after all this, how do you know when a bone sample is completely dry?
So part of it is just actually physically looking at it.
So we have them drying in a vented hood and they're in a wayboat.
So our last wash is an ethanol wash, which really helps to dry a lock quicker.
But you'll see if you go in and slip that fragment, there will still be liquid
remaining
on your wayboat.
So it's usually pretty obvious, but we give it a good half hour hour and make
sure that
it's completely dry.
And at that point too, we're dealing with a very small bone fragment.
So it's not like you're trying to dry out an entire femur bone.
We just have a small fragment that's perfectly clean with a smooth surface at
that point.
Aren't it possible to know how old the bones you're treating are?
So a lot of times as the bones, you have to go off of the information that's
been provided
to you by the investigator.
But you can also tell visually.
I will say a caveat to that is that a lot of times bone samples could be a
really fresh
sample, but maybe it's been exposed in very harsh conditions.
So it can give the appearance of being older.
When in doubt, I would just say it depends on how much sample you have, but if
you're
concerned about your quality, I maybe wouldn't go straight to rapid at that
point.
Unless you have plenty of samples to work with, then you could always try rapid
and
then move on to a demideralization, followed by an organic maybe to clean that
off as need
be.
Okay.
Now how do you prepare teeth for analysis?
For teeth, if it's going, the rapid will simply just crush the tooth and remove
any enamel.
And then the remainder of the tooth is going to go into that rapid cartridge.
If it's not going for rapid and we're going to go demideralization, then we'll
still crush
it and remove the enamel.
But then it's going to go through the mixer mill and get powdered to that fine
powder
prior to extraction.
Okay.
We have a user over here that's wondering what your experience has been Rachel
with water
logged bone samples.
You've kind of had to have some trouble using various extraction methods or
commercial
chemistries over the years.
What do you use for water logged bone samples?
Our water logged bone samples, it would depend on how long it's been in the
water.
We get a lot of small plane crashes and things like that where maybe it's only
been in the
sea for up to a few days.
I find that they actually work great.
We're lucky down in South Florida here that we have high salt water content and
things
like that.
So I find that it actually does really great at preserving the remains.
Those are samples that we would even consider going forward with rapid on.
Other samples though say it's been in water for years and years and years.
I actually also had really good luck with those as well as long as it's salt
water where
things have been a little trickier if it's in like say a acidic fog or
something like
that where it's going to be a harsher condition.
In those cases it would not attempt the rapid at all and I would go straight
forward with
demonuralization and organic.
Okay.
Now you mentioned that salt water in Florida is great for the bones.
What about temperature?
Does temperature affect the quality of DNA extracted from bone?
Yes.
Anytime you have really really high temperatures so like explosions where there
was maybe a
sustained fire with high heat for a long period of time that affects the
quality as well as
sometimes when we have samples maybe from up north that have been exposed to
multiple
freeze saws so you have their remains being frozen and then come spring they
saw out again
and then go through that cycle again for decades.
That can also have a negative effect on the quality of the sample.
Okay.
Now have you worked with an evaluated sample exposed to extreme conditions such
as disaster
victims or those recovered from seawater like you mentioned?
Yes.
I'd say a big portion of the types of remain identifications we do are from you
know,
fragmented remains from say like a plane crash or something like that so this
would be a
great option for processing those and it really depends again with anything
involving
explosions or anything like that how high the temperature was if it was a
sustained fire
for an extended period of time.
With that said though, we've gotten great results on samples that have been in
car fires for
an extended period of time too.
Sometimes it is just a lot of the draw.
Okay.
What about cremated remains?
Are you able to get a DNA profile from that?
Some of the remains are very challenging.
A lot of times too that would depend on the actual individual that was cremated
so if
you have someone with say a much higher fat content, it's very unlikely that
you're going
to have anything of substance loss as a cremated remains a lot of time.
Whereas say very thin individual you may still have bone fragments remaining.
I would say it's a incredibly challenging sample but if there are any fragments
remaining
that's definitely always something that you could look at.
I would not recommend that for rapid would be something that you would want to
go a more
sensitive route like a demeneralization to try to maximize any DNA that can be
obtained.
Right of course.
Now how many swabs do you need to take from the organs when testing?
We would just do a single swab going into the cartridge so you can always, it
depends
on how much larger piece of organ or tissue is.
You could just press into that swab and kind of roll it is how we do it to try
to absorb
some of the blood and the fluid from that tissue.
Okay wow.
Now what is your recommended approach to ideating a severe burn victim for whom
there
is no large skeletal remains like a femur, cibio, rib or so?
If there is no large skeletal remains?
A lot of times if it is burn victim first we will attempt for tissue.
So I would always ask for a piece of the psoas muscle which is kind of
protected in that
hip pocket.
So a lot of times even if there is no surface tissue you might be able to still
get tissue
from within that hip pocket.
After that really it's just what's available.
I feel like a lot of times teeth can persist in fire situations so we'll always
ask for
molars or cavity free first and then after that we would ask for a femur or any
available
lawn bone.
Gotcha.
Okay.
Last question I have where we run another time.
Did you try to use freezer meals for powder?
We did not.
We used to use blender cups and then we moved on to mixer meal.
Gotcha.
Okay.
All right audience that is about wraps up all the time we have for Q&A today.
So thank you for your participation throughout this webinar and the series.
I'd also like to take a moment to thank Thermo Fisher Scientific for sponsoring
this webinar
and a big thank you to our speakers Sherry and Rachel and especially for this
live Q&A.
This was great.
We hope that we were able to answer all of your questions.
In about 24 hours this webinar will be available on demand if you would like to
watch it again
or share it with friends and colleagues.
Additionally you will receive an email with information on how to obtain CE
credit documentation
for your participation today.
The sixth and final webinar in the Future Trends and Forensic DNA Technology
series will
be held October 15th at 8am Pacific 11am Eastern.
From this window you can scroll down in the overview section to register for
the sixth
webinar now.
If not you can also register later for DNA hit of the year 2020.
DNA database programs help solve more crime via the forensic website www.fore
nsicsmag.com.
You can also view all the other webinars in the series on demand.
Thank you so much for joining us today and we hope you have a wonderful rest of
your day.
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