Hi My name is Natalie fan and

I just graduated from Georgia Tech with
bachelor's environmental engineering and

I'm currently working in Dr average and
Dr plats that in.

Mechanical and biomedical engineering.

And today I will be talking to you about
the morphological and mechanical behavior

of fibrin clots in healthy diabetic and
sickle cell anemia and disease states.

This is just an overview of what
I'll be talking to you about today.

I'll give you a brief background about
the two diseases I'm going to address

in those being diabetes and
sickle cell disease and

I'll give you a quick
overview of the thrombosis

mechanism that occurs and
then the objectives of this experiment

the experimental protocol that we
use to obtain these objectives.

Using confocal imaging and
fiber Nala Cis and

then experimental results conclusion and
some future work.

I'm sure all of you
are familiar with diabetes.

It's very common in the U.S.

over two million people are newly
diagnosed with it every year.

There are two types of diabetes
type one which is insulin.

Deficient diabetes which occurs when your
pancreas doesn't produce enough insulin

to break down the glucose and
then type two is when

your body becomes resistant to insulin and
this is what I will be focusing on.

So essentially what
happens is your stomach

digests your food and then now you
have these glucose particles and then.

The insulin that's produced isn't being.

Your body's now become resistant to it.

So it's not responding to the insulin and
therefore your glucose is

unable to get transported into
necessary tissues and cells.

So then you have this large accumulation
of glucose in your blood stream.

The other disease that I'll be
talking about is sickle cell disease.

This is a hereditary blood
disorder that causes

the point mutation in the amino acid known
as glue to make acid and this is a hydra.

Of Phillip amino acid and
it gets replaced by baling

which is hydrophobic and
then due to this change.

We get this what's called
what's known as signalling

because the hydrophobic parts
want to aggregate together and

shield themselves away from
the water that's in your blood.

So then this sickle in
cause is a lot of problems.

Now you don't have enough oxygen being
delivered to your tissues and then

you have these blood cell conglomerations
forming which is a problem for

your blood vessels because now you have
these aggregations of cells jamming up

blood flow.

And then this is
the initiation of the human

static response which is the clotting
mechanism that occurs in your body.

So you have your normal blood vessel and

then when there's injury to the
endothelial lining of your blood vessel.

There are three main steps that occur.

Your body can starts constricting the
injured vessel that way to reduce blood

flow to that area and then platelets are
activated to create this platelet plug.

And then lastly which is the key point of

this the research I've done is
the fiber network that forms.

And then essentially you
have this blood clot.

So this is the coagulation cascade
in normal healthy conditions

the formation of fibrin which
is the network that brings in

all your platelets and your red blood
cells to form this blood clot your main.

Proteins of interest are going to be fiber
in engine thrombin and factor thirteen and

then plasma so
fibrin engine gets activated by thrombin

to form fibrin and then with
the presence of fibrin calcium and

thrombin then factor thirteen
becomes activated to come in and

form cross links in this network to
stabilise your fiber network and

then on the flip side
we have plays Minaj in

which gets activated by the presence
of fibrin and this comes in and

breaks down the fiber network when
you don't need your class anymore.

And this process is known
as fiber Nala surface.

So in normal healthy patients the thrombus
cascade is a homeostatic process.

Meaning the fiber image an activation and
fiber in polymerization

is in balance is at an equilibrium
with the fiber Nol asis.

However in these two disease states
in diabetes and sickle cell patients

have an increased risk of developing hyper
thrombotic complications such as strokes

and other cardiovascular diseases and this
is a result of an imbalance in the system.

So now we have hyper coagulation and
hypo fiber analysis.

So now in these two disease states we

are going to have an increased
concentration of fiber in engine

therefore that requires an increase in the
thrombin levels in order to politicize.

This fibrinogen the fibrin.

And then in the Lysis States.

There's going to be a decrease
in the plasma concentrations.

So in diabetes.

What happens is as I
mentioned before this.

In diabetes your blood vessels are now.

They have an increased
concentration in blood glucose.

So this glucose has
a really high affinity for

lysine residues that are present
on your cyber an engine and

plies Minaj and so what happens is this
is all floating around in your blood and

then your glucose molecules are going to
bind to all of these lysine sites and

that is going to inhibit proper
activation by thrombin and

inhibit proper cross-linking so then your
body thinks there's not enough fiber

fibrinogen being sent
to this injury site so

what it does is it'll increase
fibrin engine concentration and

that's where your hyper
coagulation factors come in and

then with plows Minaj and
it also has a bunch of lysine sites that

glucose likes to bind to so
it binds to all

these PIs Minaj insights that
are required for plasma in activation.

But now we have less PIs manage and
being activated.

So in sickle cell disease a similar

scenario occurs where we
have increased fiber and

engine concentration which then again
triggers the increase strawman generation

and then a decrease concentration of
anti-coagulation proteins like plasma.

However the mechanisms behind
these changes are unknown.

And then in addition to
the protein changes we also have

the coagulation of Sickle red blood cells.

Yes.

So the objectives of my research was to
simulate these clotting conditions under

in an in vitro state and
then to use confocal microscopy to analyze

the clot structures in these disease
states and then by using confocal

microscopy were able to maintain
the native state of these proteins and.

Cells.

And then also with confocal my
cost be we were able to determine

a fiber Narcisse rate for the diabetic.

Disease state when compared
to normal healthy patients.

So this is just a brief
overview of the protocol I

use I basically had a glass slide and
I made a chamber.

Made out of two layers
of double sided tape and

then pulling my eyes to the fiber and
clot which is just mixing the fiber

in engine the thrombin and
the factor thirteen together and then.

Covering it up.

Sealing two sides to prevent
the clot from drying out.

Yeah and then so that I use that same
protocol for all the disease states and

then for the glycated method I
would incubate the fiber engine

in a glucose solution for forty eight
hours before I pull in arise it.

And for the Sickle Cell simulation.

I would try to I put in red blood
cells and sickle red blood cells.

So for the fiber Narcisse protocol.

It was a similar method.

Except for instead of sealing
the two sides I left that open and

just let the clots and in a moist
condition that way when I was ready to

image I could just inject the plasma in
and then you would get through with like.

A camera or something to make it a uniform
distribution throughout the club.

And this is just a fast forwarded video

of the fiber knowledge service when you're
looking at it in the can focal microscope.

The action this was taken in real time and
it took about three minutes and

this was sped up to about
twenty times the speed just for

the sake of presentation purposes.

And here are some representative
images of the confocal microscopy.

Of the glycated Klotz these two images
A.M.B. show the normal class with and

without factor thirteen and
as you can see it's a home.

It's a homogenous distribution of
the fibers the pores within the clots

are larger and then the fibers are longer
as a result of proper activation and.

Cross-linking.

And then these bottom two images show
the glycated clots within without

factor thirteen and as you can see
that there's a significantly higher

fiber density and then there are smaller
and fewer pores within the clot and

then all the fiber
strands are also shorter.

Which leads us to think that there's.

The fiber imaging was not
properly activated or.

Processing.

And here the confocal microscopy
images of the sickle cell the clock

image A Here is the healthy.

Clot with normal concentrations of.

Fibrin and normal concentration
in normal red blood cells so

as you can see there is a mom.

Of the fibrin as well as
the red blood cells and

then Image be here is sickle
cell the fibrin clot with

sickle red blood cells and as you can
see the fiber network is not imagine it.

We have clusters of both the fibers and

the red blood cells and then lots of
voids where there is just nothing.

So from a fiber Nala sis
images I was able to come up

with a rate to determine the fiber
Narcisse for diabetic patients.

I just took one of the real time images
of the clots and then set an area and

then using the video I could figure
out how much time it took for

the plasma and to degrade the set area and

then I used a one way a nova and
a to keep Kramer's test with a.

P. value of point zero five to
determine statistical significance.

These are the results of that
analysis as you can see here.

Down here are the average.

Lysis rates for normal fibrin with
two hundred milligrams per mil of

plasma in which is found in
normal healthy patients and

then the fifty micrograms per mil is
what's been found in diabetic patients.

So the blue here is
the normal fibrin with.

Normal plasma in concentrations and then
that I found to be statistically different

from the results of the glycated fiber in
with fifty micrograms per mil of plasma.

However there didn't
seem to be any sort of.

Significant difference between
the normal and the Gleick a.

Fibrin with both with two hundred
micrograms per mil of plasma and

there also wasn't any difference between
the glycated fibrin with normal and.

Decreased plasma concentrations.

In conclusion the.

We found that the Sickle Cell clots
are not homogenous as evident by

a lot of red blood cell aggregation and
conglomeration of the fiber in network.

The fiber in clot structure
of diabetic clots have

were as you saw were a lot
more dense less porous and.

The fibers don't seem to have been
activated or crossed think correctly.

As a result of the altered structure and

plasma in concentrations in
diabetic patients the glycated

clots are more resistant to fiber
Nala Cis than in normal healthy patients.

And this study was unique in
the because of the experimental methods

used because again as I said
the confocal imaging allowed.

Real time.

The landing to capture real time data
while allowing the proteins in cells to

stay in their native state so they didn't
have to be fixed or anything like that.

Some future work.

I'm looking at doing is to quantify the
fiber Nala cysts of sickle cell clots and

then also to quantify the fibrin and

red blood cell distribution in normal and
sickle cell disease states.

That way we can see.

If the we can try and
see if it's the fibrin or

if it's the cells that are causing
the conglomerations and

then we also want to look at
how the R.B.C. stiffness.

Because sickle cell has different.

Cells.

So the cells have different stiffness
is than in normal healthy patients and

then how that affects the clot and
I sat should be.

I'll be happy to take any questions.

Thank you.

A lot of women would love

it while we would have to I
think we're looking at using.

Acoustic.

Like an acoustic ultrasound to
determine that and see based on.

I think how much.

Sound would pass through would and

would determine year a year density as
well as the stiffness of the blood cells.

So these are all systems

which you will be using my

only system or mimic you know what.

Yeah.

There might be so.

But yeah definitely.

I think we would definitely find.

Different results because again maybe
the flow would impact the license rate or

something but just for
the purpose of this experiment we wanted.

To create a visual and

quantify like the license rates and
then especially with sickle cell disease.

There have been very few studies
done on clotting in sickle cell.

So I wasn't even able to
find any sort of images.

Of sickle cell and clotting so
that was just but this experiment was for.

Thank you very much.

Thank.