here at the Johnson County Sheriff's Office Crime Lab, our new state of the art building here that we
have, and so what I'm going to talk to you today about is the evidence process that we go
through for DNA analysis and biological
processing So when we get a piece of evidence in,
how we're gonna look through it and see if we can find any body fluids and then eventually take those on for
DNA processing. So in the DNA section we want to be really
cognizant of the fact that we all have DNA. And we can put that DNA on to evidence
if we're not careful So we always wanna wear gloves - so always
the glove then we change in very often so we're not getting our own DNA on the
evidence which would be very bad for a case.
So when we get a piece of evidence than it
normally comes in some kind of packaging biological evidence needs to be in a
breathable type package like a paper not plastic and when it comes then it'll have a seal
here so that we know that haven't been tampered with so the officer that collects this item or
the crime scene person that collects it will put the evidence in the bag and
then seal it with evidence tape it's really easy to rip
this tape so I would know if someone had gotten into this piece of evidence
and tampered with that somehow and then will commonly see initials and
dates on these as well from whoever collected the item so it's really important when I get into
this that I do not want to break the seal either
so I can leave it intact so when this case goes to court if it
does that the officer that collected it can say yes that is my feel and it hasn't been
tampered with. So I'll open this piece of evidence in another
location. So we have these disposable razors here so we're never using the same object over again on a piece of evidence and that also prevents contamination from one piece of
evidence to another And I'll just cut the package open so that I can get the evidence out and
so here we have a white shirt gonna spread it out here so the first
thing that I'm going to talk to you about is when we get a piece of evidence such
as this in what are the steps that I would go
through to try to identify any kind of body fluids that might be on
on this So the two main body fluids that we get the
most requests for here in the lab are for blood and for semen in sexual assault cases so I'm gonna go
through those show you a few other techniques that
were used to visualize those stains and then show you some of
the tests that we use to actually see if we are in fact dealing
with blood or sement in those cases so we get a shirt and sometimes you can
visually see stains that are on it especially if it's a light color like
this is white you can see stains so we have this one
here you can kind of see here that this is a
red-brown color and so that might be a stain that could
possibly be blood and then there might be some other
stains on here but our little bit more yellow in color those could possibly be other body
fluids but we're not really sure at this point but you can visually see those so another lighting technique that we
might use if you cannot actually visualize a stain with your
naked eye is to use what's called oblique lighting so that is simply just
taking and normal flashlight in turn it on and then your lighting
whatever item you're looking at actually from the side and sometimes
what this does is that it will make a stain pop out here to your eye just slightly different a color especially
on an item where you might be dealing with a red blood
stain on a red cloth or on a darker type item don't like black
or brown just that little bit different in side
lighting will make that thing pop out to you see you can
visualize it almost like it wet in appearance at this point anything
that you were able to see visually either with your naked eye are using the
oblique lighting I'll go ahead and marked those right now
as possible stains to test later on either for our blood or semen
test depending so since this stain that we looked at
earlier is red brown in color we're probably gonna test that for blood later on so how that I'll go ahead
and mark it is I'll identify the stain and then I'll
just take sharpy and start just very close to the
stain encircle at and then I'll give it and identifier
each each stain will have its own unique identifier so for myself
with bloodstains normally I will letter those so A through Z. With semen stains are usually number
those in chronological order so since this is
my first stain I'm going to call it Stain A and I'll go
ahead and just write an A right next to it so I have my stains identified for
testing the the last resort that we can use for
trying to identify stains is by using what's known as an alternate
light source or a ALS so this is our alternate light
source right here and what it does is it has lights the different wavelength on it and
for biological fluids stains we're looking at wave link between 415 and 500
nanometers of light it is not the visual light that you can
just see with your naked eye and so what it does is we shine that
light onto whatever item that evidence we're looking for and then by using a filter either in the
form of glasses such as these or a filter plate that you can put over camera if you
would like to take pictures of what you're looking at it'll filter out all light except for
what is fluorescing from the body fluid so you'll actually be able to see stains
that you could not visualized with your naked eye especially again on darker colored
clothes which I'll show you the difference of what a stain looks like under the ALS
from this white shirt to this black pair of shorts The ALS has to
be used complete darkness so we make sure that things like windows
have the blinds covered so that the room can be
completely dark so what I'm gonna do is turn off the
light and then I'll have these filter goggles on for myself to be
able to see any stains that we visualize you can see how easy it was to see the
stains on the nice light, white shirt that we have
unfortunately its a little bit more difficult when you
have a darker type fabric like this black so when we're searching something like this we might try different wavelengths of light on the ALS t to see if we are
visualizing any stains or not and then when we have a fabric
that;s dark like this we are probably going to use something
like a silver sharpy that would actually show up on
the black fabric instead of using a black marker which won't show up that'll help us identify which stains
were going to test something to realize about the ALS is
that when we're looking for stains with alternate light source anything that you see that fluoresces
under that light we're not really sure what the source of
that stain is every body fluid except for blood will
fluoresce so semen saliva sweat urine all those
will fluoresce an then also things like soda pop and other
fluids might fluoresce under the light to you so we're simply
searching item very quickly to try to identify any stains that we want
to take on to further test for at the different body fluid test
that we had a lab and the last thing I.
Wanted to show you is on something like this so this is a piece and drywall that we
actually we can cut this out at the crime scene
if we have to to preserve bloodstains so on something like this we are able to see the bloodstains very
easily that's no problem they're dark red stains so we don't really have a problem
visualizing them something we might do you wanna piece of evidence like this is actually
determine what kind of patterns of bloodstains that we have and are there any differences so on this be
the drywall were actually think you different kinds of patterns these two here look more like contact or transfer pattern maybe
somebody wiped their hand across it just looks
more like a a smudge or a wipe and then we have all these
little stains here that look like they came from blood
actually flying through the air and hitting this drywall so for instance
like this big stain down here or these
smaller ones they all look like they could have come
from blood flyint through the air and then hitting this wall so how we would differentiate these is to call them two different patterns
pattern A. Might be the wipe stains and then pattern
B. Would be these circular stains that may have been somebody at being hit in
the head some kind impact that may have created those those type stains and will test those separately for DNA. Maybe thinking that those possibly
could have come from two different people we don't know but we would differentiate
them that way so that we could no what DNA came from which pattern So the first tests were gonna do is the phenolphthalein test we take one drop a sterile water onto a
sterile swab that's enough to pick up any blood that we wanna test.
You don't
need very much blood on the swab just enough to be able to see a stain then we're gonna add one drop of the phenolphthalein one or two and one drop the hydrogen peroxide and it should turn
a nice bright pink color very fast within 10 seconds and that means that we have a positive
test gives us a good indication that we're
dealing with blood. The next blood test that we can do you is called him a hemotrace test and this also detects hemoglobin but it specifically
detects human hemoglobin so it's supposed to be a human
specific test to tell us if we are dealing with human
blood however this again is a presumptive test and there
are higher primates such as monkeys and ferrets that react with this test as
well so it's not a hundred percent but it still gives us really
good indication that most times we should be dealing with human blood So this tests actually fairly resembles a
pregnancy test it's very simple to use what I would do is take the blood that I've swabbed up and add it to this buffer and we let it
sit there for 10 minutes and then I'll add it to this card and
for our purposes today I already have one that I've already extracted so
there's a blood sample in this vile so I'll open this up fill my pipette full of the and now extracted
blood and just add it to the end to this
cartridge and much like a pregnancy test if you
get two lines it's a positive reaction and the only get one its a negative
reaction for human blood and this test takes a few minutes to run if you see a positive result immediately
then it's positive but to call it negative you would wanna
wait a full ten minutes and make sure that there was no second
line that formed on the card This card has only run for about a
minute and we can already see that there are 2 lines there control line is on the top which means
that the test is working and the test line is the bottom line which is whether it is going to be positive or negative and so we do not need to wait a whole 10
minutes for this and since we are already seeing a positive reaction if it was negative and we were not
seeing that test line we would wait a full 10 minutes to make sure that it didn't
show up. Once we see this positive we can take
a picture of it and we'll definitely document in our notes
that it was a positive test unfortunately if you let these that much
path about half an hour you might get a false positive starting
to show up so we don't actually keep these as evidence because of that reason but
we will take a picture or have it in our notes that it was positive. Sometimes when we are out at a crime scene we might be looking for blood but we don't get those nice dark red
brown stains that are really easy to see either from somebody that has cleaned up a
crime scene or you just get really diluted stains
and or you might be in an area like an office building where something like the carpet is really
hiding those stains because that's what it was meant to do so there are times when you're looking
for blood that we're not really able to see with our naked eye very well are that may be worth searching for
patterns such as footprints leading somewhere are
just other patterns were trying to pull out so in that case for any use a chemical
that is called luminol and what this does is it works with the
hemoglobin and blood as well and if there's hemoglobin present it
excites the luminol to a state that it does not like to be an and so to get back to luminol's grounded state it emits a light blue fluorescence and so if
blood is present we will start to see a light blue fluorescence appear now this is again a presumptive test
that we just used to look for patterns and other things can make the luminol flouresce such as bleach or other cleaning materials so sometimes when cleanup is an issue it
might pose a problem but during this demonstration our show you what bleach
might look at what look like when we use the luminol.
So first I'm gonnal make the luninol reagent and the chemicals that are used are
sodium hydroxide hydrogen peroxide and then luminol
itself and water so we're gonna do a ratio of seven parts water to one part each
of these other chemicals so here I have 350 microliters or milliliters of water and we're going to put that into are
spray container because we're gonna spray this chemical
onto whatever surface that we're testing and then we're gonna take 50 milliliters of our sodium hydroxide and it doesn't need to be extremely
precise but as close as you can get it 7 to 1 to 1 to 1. 50 Milliliters of the hydrogen peroxide and then finally 50 milliliters of luminol and this is in a brown bottle because
it's very sensitive to light so we try to keep it closed off to light and for this reaction you have to be and a completely darkened room or if this
is something you're searching for outside you're gonna have to wait till night
time when it's completely dark and then possibly hope that there's no street lights or
anything else that would hinder the process because it really
does need to be pretty dark to be able to see this so kinda makes it
up. This is the sheet I prepared to use
luminol on and let's pretend like this is actually
a floor at a crime scene that you're trying to see any blood patterns that might be there
maybe you think somebody cleaned up and if you can see here there's a kinda diluted red stain right there and I can kinda see okay maybe it's red
it might be blood I don't know but maybe there's some kinda pattern
there that the luminol can help enhance our bring to light so at this point we would need to make
the room completely dark still makes a return of all a light and
then we're gonna spray our luminol re-agent over the entire area and if we have a
positive reaction with blood it'll give a light blue fluorescence
that'll start to glow and it'll start to fade and then after we see the pattern that have been
placed on this at test demo then I'm going to spray
bleach solution on it and show you what it
looks like if you were to actually be getting a reaction from something like
bleach or a chemical cleanup type of a chemical. Ok so we will turn off the light (sound of chemical being sprayed) let me show you what it's like if you
were to have bleach or another chemical that's reacting you gonna get kind of a sparkly looking
reaction So you see how that sparkles and then fades that is more on the reaction that you
would get with a bleach type substance the next set of tests that I'm going to do
you are that three tests that we have to identify semen and being in the DNA section in
the laboratory we get a lot of sexual assault and this
is the main body for that we're going to be looking for in a sexual assault so the first half but I'm going to do.
Is called the acid phosphatase test it is a
presumptive test very quick and it's actually testing for
it an enzyme that's found in seminal fluid its found in other body fluids as well
which is why it's presumptive but it found about 20 to 300 times more
concentrated in seminal fluid than any other body
fluid so it's a pretty good indicator that we could be possibly be dealing
with semen so this test that first thing I'll try is to take a sterile swab again and I'm
going in moisten it with just a drop of sterile water and
I'm going to attempt to swab the stain sometimes on clothing the the cloth does
not wanna let go of the fluid that attached to
the cloth so I'm gonna try it and we'll see if
it gives us a good reaction here sometimes you have to swab pretty forcfully to pick up the fluid when when you're dealing with cloth and this is our acid phosphatase chemical here and what we're looking for we'll drop it onto the swab and what
we're looking for is a pink to purple reaction on the swab now to give us an indication that we are dealing with semen so here's
a drop you can do 1 to 2 and this
reaction takes up to thirty seconds to react so it's
not necessarily quick but we're looking for a pink to purple reaction on the
swap and so I'll wait a full thirty seconds
before calling it negative if it doesn't turn pink but if it does t urn pink then we have a
positive reaction you can kind of start to see this pink
purple color forming here on that very tip of the swab and so that's a pretty good
reaction there I would call that positive and just notate that in my
notes that that was positive another thing you might do if
you're suspecting that a stain is not being able to be swabbed
up is you can actually take a cutting of
the stain and apply the reagent straight to the cutting.
I;m gonna
take a disposable razor again we do not like
to reuse items here in the crime lab and I'll just take a little cutting inside of my stain place it onto a separate piece a paper
clean piece the paper and then I'm going to apply the acid phosphatase reagent straight to
that cloth and might take a couple of drops to get it soaked in there and might use a little tooth back here
to help me get the chemical soaked in there and you can
start to see this line here where we're getting a nice dark purple reaction and again I'll take
up to 30 seconds so you can see now how it's turning purple and it'll just get darker and
darker as it goes but that get this is giving us a much
darker reaction and the swab because since we're directly testing the item you're
getting all that enzyme your testing for is all soaked into the cloth there we have a second at presumptive test that's
called the P-30 test it's a test for prostate specific
antigen or PSA that is found again in seminal fluid however like many of our tests PSA. Can be found in other body fluids such
as breast milk so it is still a presumptive test and the P-30 test is very similar to the
hemotrace test where it's just the cartridge that looks
like pregnancy test almost exactly the same and it works in the same way as the
antigen-antibody reaction and what we would do is take another cutting and place it directly into a tube and then we're going to add a PBS buffer to the two about 500 microliters or half a milliliter that's about right and then this we would vortex which means to shake
it up on our vortex and then let it incubate for 10 minutes
to two hours to let that semen that possibly in a stain extract
out into the liquid and for purposes other video I have one here that I had let extract
for few hours. 10 Minutes is plenty but you
can let it go for sake of convenience and this we now see that may be that the
caller has changed here in a liquid and extracted out into there so what we
would do is take the piece of cloth that's been
extracting and we have a little basket here with holes in the bottom we would take this take a toothpick pull out that cloth that's been
extracting and put it into the basket and then put the basket straight into
the tube here and close it up and now this we would put into a centrifuge let it spin
down for three minutes to pull all the liquid out of the cloth
that was in there and to also pellet any sperm cells that
might have extracted into the liquid to the very bottom up the tube so then ideally we would end up with is any
enzymes from the seminal fluid in the liquid and then sperm cells that are intact
pelleted at the very bottom. So now that we've sent centrifuged this down
we can open it up and discard this piece because theoretically
there's nothing left on that piece of cloth and now we're left with this liquid and then a
sperm cell pellet in the bottom so what we can do is a few things we can take almost all the liquid here out of the tube leave just a very small amount in the
bottom to use for our sperm slide and what I'll
do is put half this liquid into the P-30
cartridge and then I'll put half of the liquid into
a clear test tube here this is the third way you can't ask for that AP test is by
using this extract now I can add the AP reagent
that we used before a couple drops into the test you and we can make sure
again that this is turning now a nice bright pink or purple color and this is really nice because you're
not dealing with any fabric so you're not having any colors that
mess around with your reaction you can see a nice dark purple there that is a really good indication that you're
dealing with semen and that can just get thrown away and then this P-30 cartridge much like the hemotrace will also
run for 10 minutes to call it negative but if you see the two lines for it that
will be a positive reaction and you can just call it positive from that point
exactly like we showed you with the hemotrace card you can see here you can see a faint
line there in the test and area and it only get darker but right
now that is positive.
The third and final Test that we will do for semen is actually a microscopic
sperm cell search and this is the only confirmatory test
that we have for semen if you see a sperm cell that is a hundred percent positive for
semen being present on that item however we do deal with cases where
we have males that maybe have gone through a vasectomy and do not have any sperm cells left and so using the two
other tests that I showed you our two presumptive tests together gives us an indication that
semen is present without seeing sperm cells so that body fluids
that react with the acid phosphatase test that are not semen in are not the
same body fluids that react with the P-30 tests that are not semen so we can pretty conclusively say that
if we get a positive result on both those presumptive tests that that's a good indication that we're
dealing with semen and maybe we have somebody who's been vasectomized or somebody with a really low sperm
count and we're just not seeing sperm cells on the slide So I'm gonna show you how we make are sperm
slides and we're going to take the remaining
portion here that was left in our tube that we extracted and hope we have a
sperm cell pellet in the bottom. Now we're ready to centrifuge this down
down this is our centrifuge we put our sample in one side
and then we always want to make sure that this is balanced and so we will put a blank sample on the
other side to balance it out as its turning Put on the lid and we'll let it go for three
minutes (timer buzz) now we should have our sperm pellet. To start making our sperm slide we want
to take the remainder this liquid pipette up and
down a few times to get that sperm cell pellet broken up and then
we'll go ahead and put it on a microscope slide that's on a hot plate set at 60 degrees
Celsius and it'll make a nice little dried stain we let it heat
up in heat fix to this microscope fight for 20 min so after the stain is heat fixed to
the slide we're going to stain it with what's
called a Christmas tree stain it's called Christmas tree because it's one
red stain and one green stain and the way the stain works is that the
red stain will will stain the sperm cell head it'll
stain half of it red and half of it pink and then the green stain will stain
the sperm tail and also a stain any skin cells that are
there the outer portion the cell will be green the
nucleus of the cell will be red So the first thing that i'm gonna put
on is the red stain you just need enough stain the cover where you put your sperm
cell pellet and that sits for five minutes after the five-minutes is over you wash it off with sterile water and then you apply the green stain for
only 15 seconds and again after the 15 seconds just wash
it off and now your microscope slide can sit here
and dry and when it' s dry it's ready to go onto our microscope to search for sperm
cells. Here we are with our sperm slide and this is one of our microscopes here in the lab this is has a dual function it is a
polarizing light microscope and it also has a few filters here and
for a different fluorescent dye that we can
also use to search for sperm but for our sake today with the
Christmas tree stain we don't need to use those filters but it does have that function
so I'm gonna put this slide right here on the table the microscope and then I need to look in the oculars here and get the slide focused to where I can
see what's on there and then start searching it to try to
tell if I actually have sperm cells which will be stained a red color So I'm gonna look in here and I'm gonna move the slide so I am focused where I know there's
staining now I'm going to focus in on the slide and see if I
see any sperm cells which right away I do there's a lot of sperm on this slide.So I'm going to get it right focused in the middle here and then this
microscope also has the capability also to being able to
to view in oculars and on the computer at the same time
The occulars are always gonna give you the best view but if you're wanting to take a
picture i what you're seeing then it's really nice to have the
computer view so that we can snap a picture and keep that in a case file especially
if we have a case where we're only seen one sperm cell on the whole entire slide that still gives us enough evidence to know
that we have a semen stain because we are seeing that sperm cell its very
sensitive but when we only have one it's hard to just say in your notes I
just have one sperm cell so it's really nice to have a picture of that cell so I'm gonna move the
microscope to wear it half viewed on the screen in half viewed on
the microscope and so you can see here this is the same
view that I would be able to see out in the eyes and the microscope and
it's set to be I an ideal color balance setting for the staining that we use and all
these little red spots here are sperm cells and so you can see that the top with a sperm cell is kind of a light
pink in the rest of it is red and then sometimes we will see a green
tail on the sperm cell itself we do not need a
tail on the sperm to call sperm cell all we need is that double
stained head and so what I'll do at this point I
can come in here and I can capture the image so it'll capture it down here and then I can save that image to be able to pull up where are
put into our case file into this would be an example of an
image that's been saved we can attach it in our notes or we can print it out and put it with
their report whatever we need to do and so this is a really good and view
of a lot of sperm this would be what we would call a 4+
sperm or the highest number of sperm that we would see on a slide so it just means that there was a lot of them in there on that
sample there sometimes when we might see nothing and then just one sperm cell and that's it.
Or I have another example here to show you this is an example of what we might see when you have skin cells mixed with the
sperm cells which is something we would see on something like a swab from a rape kit that was taken
from a a female's body and then also has sperm cells on it so this cell right here as well we call
epithelial cells and that a skin cell and so here we've
got the green staining pretty large there with a red nucleus
not just a normal skin cell in the nucleus is much larger than a
sperm would be and then here there and there are three sperm on this
slide as well so we have a few epithelial cells and three sperm and so we can say that not only do we
have sperm there but we also have some kind of skin cells we don't know who they came from they
could come from the male or they could come from the female in this case and then we'll bring it on to DNA testing. (Music) (music).
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