Microcirculation capture by Microscan

2008-2010

Gabriel Najarro, Nathan Shapiro, MD

 Microcirculation capture by Microscan

Introduction
Welcome and we thank you for participating in the NIH sponsored study, “Endothelial Cell
Signaling and Microcirculatory Flow in Severe Sepsis.” This investigation is an ancillary study to
the Protocolized Care in Severe Sepsis (ProCESS) trial. The current project will utilize 9 of the
ProCESS sites to extend the intellectual reach of ProCESS by intensively investigating the role of the
endothelium in sepsis, as well as by studying microcirculatory flow disturbances.

There are two main aspects to the current study:

1. Endothelial Cell Signaling
The first aspect is to investigate how the endothelium responds to sepsis and sepsis therapies. To
accomplish this, we will use biomarkers as our primary readout, grouped into the categories of
endothelial cell adhesion, coagulation, and vascular endothelial cell growth factor (VEGF) signaling.
As part of this initiative, we will attempt to develop a biomarker panel that is predictive of subsequent
organ dysfunction and mortality.

2. Microcirculatory Flow
The second main aspect to the study is microcirculatory flow. The microcirculation is defined as the
smallest vessels (≤ 100 µm diameter) where gas and nutrient exchange takes place. Previous data
suggest that disturbances in the microcirculation play a crucial role in the pathophysiology of sepsis.
If sustained microcirculatory dysfunction is present, it can lead to respiratory distress in tissue cells
and subsequent organ failure, even in the absence of global hemodynamic deficiency. We will use the
Microscan device which is a videomicroscope that is able to magnify and record blood flow from
under the tongue to aquire images of microcirculatory flow. Using these images as a
representation of flow disturbances, we will investigate the role of the microcirculation in sepsis.

On behalf of the ancillary study investigators and the ProCESS investigators, I would like to thank you
in advance for your participation.

Sincerely,

Nathan I. Shapiro, MD, MPH (Principal Investigator) on behalf of the Investigative team

Ancillary Study Co-investigators and Collaborators:

William Aird, MD Long Ngo, PhD John Kellum, MD Derek C. Angus, MD, MPH
Don Yealy, MD C. Ince, PhD Stephen W. Trzeciak, MD

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Table of Contents
Background 4

What we measure 6

Hardware 7

Software - Capture 10

Software - Upload Manager 11

Area of Interest 12

Bedside Use 13

Image Quality 16

FAQ 19

References 20

Quick Start Guide 21

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Background

Why is the microcirculation important?

The term “shock” has been previously defined as inadequate O2 supply to meet the metabolic
needs of cells or organs. However, the story is more complex as mechanisms of O2 supply
(macrocirculatory flow), distribution (microcirculatory flow), and processing (mitochondrial function)
must all be functional and adequate to maintain normal physiology. Accordingly, the potential areas of
deficiency may be broadly classified into three types of failure: 1) macrocirculatory, 2)
microcirculatory, and, 3) mitochondrial failure. Macrocirculatory failure is assessed through global
parameters such as mean arterial pressure, cardiac index and mixed venous oxygen saturation
where a deficiency results in an inadequacy in the net amount of O2 being sent to the tissues.
Microcirculatory failure occurs with physiologic shunting or maldistributed flow resulting from
disrupted perfusion in the small arterioles and capillaries. In some instances there may be adequate
macrocirculatory flow, but inadequate mircocirculatory perfusion. Finally, mitochondrial failure occurs
when the mitochondria are dysfunctional and unable to process the O2.

The causes of microcirculatory flow disturbances in sepsis are multifactorial and include
endothelial cell dysfunction, increased leukocyte adhesion, microthrombi formation, rheological
abnormalities, altered local perfusion pressures due to regional redistribution of blood flow, and
functional shunting. These derangements can cause marked alterations of oxygen transport including
impaired tissue oxygen extraction. With the advent of new imaging modalities such as Orthogonal
Polarization Microscopy (OPS) videomicroscopy and Sidestream Darkfield (SDF) imaging, it is now
possible to visualize the microcirculation in human subjects. Prior studies demonstrated that
persistent microcirculatory alterations refractory to resuscitation are highly prognostic of fatal
outcome, independent of systemic variables and oxygen derived variables. Given the critical role of
the microcirculation and its endothelial cell lining in mediating flow and oxygen delivery, OPS/SDF
has the potential to provide unique and important diagnostic information in patients with sepsis.

The Microscan permits direct visualization of blood flow in the sublingual microcirculatory network
in human subjects in a non-invasive fashion using a hand-held videomicroscope. The technique has
been validated in both experimental and human studies. Why the sublingual site? Numerous
investigators have demonstrated that impaired sublingual perfusion can track impairment of
splanchnic perfusion and detect early systemic perfusion failure in shock states. Monitoring sublingual
blood flow can yield important information for use in clinical studies of circulatory shock because (1)
the sublingual mucosa shares the same embryologic (and therefore anatomic) origin as the
splanchnic mucosa, (2) derangements in sublingual perfusion reflect derangements in splanchnic
blood flow and (3) the sublingual space is easily accessible. Because splanchnic hypoperfusion is
one of the earliest indicators of systemic hypoperfusion in circulatory shock, impaired sublingual
blood flow can herald the onset of systemic hypoperfusion.

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Background

What do we record?
• Using the handheld video microscope
Microscan we can record realtime blood flow in
the smallest of blood vessels. We typically
record 15-20 seconds of a single field of view
while maximizing focus and minimizing
pressure artifacts. We repeat the imaging for
at least 3 different fields in the sublingual
space. These video typically yield
visualizations of arteriole, venule, and capillary
blood flow.

Microscan
How it works
•The videos are captured using a combination
of a Charge Coupled Device (CCD) camera as
a sensor, 5x lens for magnification, and strobed
green LED for illumination. As green light
passes through the sublingual tissue some is
absorbed by red blood cells while most is
scattered back towards the camera. The light is
then magnified and detected by the CCD which
produces an image where dark areas are
representative of blood and white areas are
representative of light scattering tissue.
•Each pixel in the image represents 1.4 um.
Typically capillaries and red blood cells are
5-10um in size. We can effectively see as small
as 4.2 um.

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What we measure
First, from the 1-2 minute clips, we edit it down to 10-15 seconds of representative video that is
optimal for analysis. Optimal videos are: representative of vessels, stable, focused, have good
contrast, and are absent of pressure artifacts (see Image Quality section). Then we score the videos
by characterizing the flow in the smallest vessels (<100µm & <20µm) – there are a number of
possible scoring systems:

1. Flow – We assign values to each vessel in the image based on the type of flow (none - 0,
sluggish - 1, intermittent - 2, continuous - 3) and calculate a mean flow index. This will represent
any alterations to microcirculatory perfusion due to inflammation, coagulation, decreased
fibrinolysis, or endothelial injury.

2. Density - We calculate the total area occupied by vessels and divide it by the total area within the
field of view to obtain the density of vessels. Any changes in density might represent a
reorganization of blood flow due to alterations in microcirculation or the disappearance of vessels
due to occluded flow.

3. Functional Capillary Density - We calculate the total area occupied by flowing vessels and
divide it by the total area within the field of view to obtain the density of functional vessels.

4. Heterogeneity - We measure different flow rates within a single field of view.

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Hardware

Microscan Head
1. Light Intensity
2. Tip Ejector
3. Video Connector
4. Indicator LED
5. Focus
6. Removable Handle

Microscan Battery Unit

1. Power
2. Charger Connection
3. Video Connector from
Microscan Head
4. Video Connector to ADVC
5. Power LED
6. Charge LED

Extras
1. Microscan Imaging Head
2. Spatial Calibration Unit
3. Removable Handle
4. Battery Unit
5. Power Plug
6. AC Adapter
7. Disposable Tips

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Hardware
Analog to Digital Video Converter (ADVC)
Signal Selector Video Output Power
Video Input

Front Back

Notes about the Hardware

• Tips are a limited resource. Be sure to have all other hardware setup before opening the sterile
package so as not to waste tips.
• IMPORTANT - Keep the Microscan probe tip covered by a designated protection tip at all times.
Reuse this tip whenever storing the Microscan head. Never leave the tip uncovered as you risk
scratching the front lens.
• Microscan runs on batteries! When not using the Microscan Unit keep it plugged into the wall with
the AC adapter so as to always have a full charge
• Make sure “Analog In” is selected on the ADVC when connecting to the laptop. This makes sure
the analog signal coming from the Microscan head gets converted to digital in the ADVC.
• When using the Microscan do not keep the Battery Unit plugged into the wall, it has a trip switch
which keeps power from being sent to the Microscan Head to power the LED’s and internal CCD.

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Connection Diagram

Connecting the Microscan to the Computer

1 1. Connect gray cable A to Microscan Head.
2. Connect gray cable A to Microscan Battery
Unit.
Cable A 3. Connect black cable B to Microscan Battery
Microscan Head
Unit “Video Out”.
4. Connect black cable B to front ADVC “Video
In”.
5. Connect 6 pin end of Cable C to back of
ADVC
6. Plug in the appropriate power adapter into the
back of the ADVC
7. Connect the 4 pin end of Cable C to the
Laptop
8. Remember to activate the Signal Selector on
Cable B
the ADVC once everything is connected and
powered on
Battery Unit
2 3

Front

5 6

Back
ADVC Cable C

6 pin end

4 pin end

Laptop

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Software - Capture
The software we use to capture videos 1
is called Microscan by Study Maker 3
and is very straightforward to use. 4 2
1. Maximize the window so as to
utilize the entire screen.
2. Input Patient #, Time Point, Site ID,
and Operator.
3. Once you’re ready you can being
Capturing by clicking the “Capture”
button on screen or press C on
keyboard.
4. Once you’re satisfied with the video
you can hit the “Stop” button on
screen or press C on keyboard.

5. If you right click on the thumbnail of a video you

can activate some extra options
6. You can play the scan to review it for quality
7. If the video contains excessive movement,
5 bubbles, cloudy saliva, blank space etc. You
can delete the scan. *NOTE* this will
6 permanently delete the scan so be careful
7 8. Use the comments section to make any notable
8 characteristics about a particular scan.

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Software - Upload Manager

2
1. Acivate your laptop’s connection to the
internet
2. You can toggle between the Scan/
Capture Manager and the Upload
Manager with this button
3
3. Enter your email address for 4
identification purposes
4. Click Enter & Proceed

5.You should have a list of all the files

pending upload
6 6.Once you have the laptop plugged into
your hospitals network you can click
“Upload Pending Files” to start
uploading.

7. At this point another smaller Internet

Explorer window should open while
the files are uploading
8. Do not close this secondary window
until all the files have completely
uploaded
7

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Area of Interest
In the diagram you can see the highlighted area
underneath the tongue which will yield the best images.
Ideally we would like one set of data from each of the
numbered areas.
These areas have the the thinnest layers of epithelium
allowing the Microscan to illuminate the blood vessels
optimally.
Further more, scans obtained anterior to the plica
sublinguas (4) tend to be more stable with less random
drift than posterior to the Plica Sublingualis.
The easiest way to see this area is to have the patient
1 3
curl their tongue towards the back of their mouth. Once 2
you’ve advanced the probe to a desirable area you can
have them rest their tounge in either a comfortable
position, against the front of their palate, or to the outside
of their left or right upper teeth.

4
4

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Bedside Use
Obtaining good images is a challenge. Essentially the operator is holding a microscope freehand
(so all movement is magnified). What needs to occur is the patient needs to stay still, the operator
needs to stay still, the camera needs to be still - so the resulting image is still.
Furthermore, you can't push too hard or you will occlude flow, and you can't push too soft or you
will lose contact with the surface. Finally, focus and light need to be adjusted. Challenging? Yes!
Answer, like most things, knowledge and PRACTICE! PRACTICE! PRACTICE!

Positioning: Method 1
1. Stand on the same side of the patient as your dominate hand (i.e. If you are right handed stand on
the patient’s right side)
2. Place the microscan laptop on the opposite side of the bed in a direct line of site.
3. Have patient rest their head against the bed.
4. Raise the bed to a height where you don’t have to bend over excessively.
5. Have the patient use 2x2 gauze to dry out any saliva/secretions underneath their tongue regularly
throughout the scans.
6. Attach a new plastic tip to the Mircoscan probe making sure it clicks into place and the light on the
back of the Microscan goes from red to green as secures into place. If the tip isn’t attached
completely the images will never focus.
7. Brace yourself with your non-dominant elbow against the bed
8. Use your non-dominant hand to stabilize your dominant hand between holding the Microscan
device and the patient’s chin.

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Bedside Use
Positioning: Method 2
1. Stand at the head of the bed behind the patient.
2. Position the microscan laptop so that you can see both it and the patients mouth without turning
your head too much.
3. Have the patient rest their head against the bed
4. Raise the head of the bed so that you can see easily into the patients mouth.
5. Have the patient use gauze to dry out any saliva/secretions underneath their tongue regularly
through out the scans.
6. Make sure to use a new probe tip and that it is attached properly to the Microscan head.
7. Brace both of your elbows against the head of the bed.
8. Plant your dominant hand holding the microscan device against the patient’s cheek for
stabilization. You can also rest the Microscan against the patients upper teeth for extra
stabilization.
9. Use your non dominant hand for focusing.

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Bedside Use
Obtaining Images
1. Have the patient open their mouth to a comfortable position, keeping the mouth open too wide is
detrimental to image acquisition.
2. Gently insert the probe tip into the patient’s mouth. Have patient gently rest their tongue either
against their palette or along the top of the probe.
3. Depending on your angle rest the probe
against their upper or lower teeth for stability
4. Gently advance the probe into the sublingual
area until the flow is partially or completely
occluded.
5. Retract the probe from the sublingual
mucosal surface until contract with the tissue
is lost
6. Just before contact is lost, you will see what
flow looks like with no pressure. This
represents an acceptable image quality for
recording data
7. Advance the probe again slowly until contact
is regained and the microcirculation comes
into view.
8. Focus the image.
9. Once you have a stable image start Capture
on the software.

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Image Quality
Obtaining good videos with the Microscan device requires some consideration for the following
factors:

1. Pressure - Simply put - if you push too hard, it will occlude vessels
and cause a false representation of slow flow. THIS IS THE MOST
COMMON CAUSE OF POOR IMAGE ACQUISITION!!! And,
unfortunately perhaps the most challenging problem to fix. The
larger vessels (veins and venules) are thin walled and will
occlude first. They are your indicators - if you have good flow
in the large vessels, you are not pushing too hard. You should
go to your area, focus, then lighten pressure until you lose contact,
then slowly advance. You should look for a point with maximal flow,
including flow in the large vessels as your indicator that you are not
pushing too hard.

2. Focus - The Microscan head has a focus knob which changes the
distance between the camera and the lens within the head, thus changing the focal plane. It’s
important to use this knob to search for the best plane of focus when imaging. A second person
focusing can be helpful, otherwise you will need to get accustomed to holding the scope with one
hand and using your thumb to focus.

3. Illumination - Make sure that you adjust the amount of light illuminating the sample by using the
“Light” knob on the back of the Microscan head. Too little light and we won’t be able to visualize
all the vessels in the field of view. Too much light and the image will appear washed out. If the
field is initially blank, it is often because the light is not adjusted correctly.

4. Movement - Both patient and microscan operator need to hold still. It is a challenge as the
images will “magnify” any movement. Please try to obtain images that are as still as possible.

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Image Quality
Good Image Traits
• Illumination is sufficient to visualize vessels. In
these image we are able to see very small
vessels clearly.
• Fast consistent flow in the large vessels.
• Small vessels are focused well.
• Contains different sizes of vessels.

• Most vessels donʼt loop onto themselves (causes

false representation of fast flow, see examples
on next page)
• Overall the video maintains the same field of
view for 10-20 seconds.
• The background is illuminated relatively evenly -
the amount of gray in the non-vessel spaces is
the same throughout the image.

• All the larger vessels show no signs of pressure.

If you apply slight pressure to the tissue and pull
back you should see the flow in those larger
vessels slow down and then speed up again.
• We donʼt see excessive image degradation from
saliva.
• No parts of the image are being blocked my
bubbles.

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Image Quality
Bad Image Traits
• Illumination is to low, small vessels are not
easily visualized.
• Too many vessels out of focus.

•Bubbles are blocking some of the image.

•Vessels are out of focus.

• To many out of focus vessels. Looped vessels

• Background illumination is uneven.
• Too many looped vessels.

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FAQ
Why don’t I see any video on the computer?
Most likely the “Signal Selector” button needs to be activated, select “Analog In” on the ADVC. If
that doesn’t work make sure to double check all of your connections.

Why is there a red light on the back of the Microscan unit?

The light stays red until you have attached a tip and it clicks into place. This is important to get a
focused image.

Why is there a flashing yellow light on the Battery Unit

This most likely means that the Battery Unit is out of power. It is important to keep the unit
plugged into the wall when not in use.

There never seems to be fast flow, is it something I’m doing incorrectly?

This is most likely due to the user applying too much pressure. Make sure to pull back from the
tissue enough that you can begin to see fast flow in the large vessels.

What is pressure artifact and how can I avoid it?

Pressure artifact is when you push too hard, falsely occluding the flow. The larger venules are
thin walled and will occlude first – these are your indicators. If there is good flow through the large
venules, you have a likely have the right amount of pressure. If flow is occluded or intermittent, you
are probably pushing too hard – try to lighten pressure. If the speed of flow increases – you were
pushing too hard and need to balance your pressure to obtain the fastest flow possible.
Pressure artifacts can be seen in the entire field of view or only parts of the field of view. It’s
important to watch for it in all areas of the image.

What if the microscan battery back is still plugged in?

It simply will not work. The cover door on battery pack that blocks the plug for the AC adapter has
a safety mechanism that does not allow the camera to be on while the door is open. Make sure to
unplug the battery pack when you are ready to start scanning a patient.

What happens if the screen is all white?

This is usually because the light setting on the microscan head is set to high. This results in over
exposed images. Reach to the back of the microscan head and turn it down.

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References
Want to learn more about the microcirculation?

Here’s some selected references:

Review Articles
Bateman, R. M., M. D. Sharpe, et al. (2003). "Bench-to-bedside review: microvascular dysfunction in sepsis--
hemodynamics, oxygen transport, and nitric oxide." Crit Care 7(5): 359-73.

De Backer, D., J. Creteur, et al. (2002). "Microvascular blood flow is altered in patients with sepsis." Am J
Respir Crit Care Med 166(1): 98-104.

De Backer, D., S. Hollenberg, et al. (2007). "How to evaluate the microcirculation: report of a round table
conference." Crit Care 11(5): R101.

Ince, C. (2005). "The microcirculation is the motor of sepsis." Crit Care 9 Suppl 4: S13-9.

Spronk, P. E., D. F. Zandstra, et al. (2004). "Bench-to-bedside review: sepsis is a disease of the
microcirculation." Crit Care 8(6): 462-8.

Trzeciak, S., I. Cinel, et al. (2008). "Resuscitating the microcirculation in sepsis: the central role of nitric oxide,
emerging concepts for novel therapies, and challenges for clinical trials." Acad Emerg Med 15(5): 399-413.

Trzeciak, S. and E. P. Rivers (2005). "Clinical manifestations of disordered microcirculatory perfusion in severe
sepsis." Crit Care 9 Suppl 4: S20-6.

Original Research
De Backer, D., J. Creteur, et al. (2006). "The effects of dobutamine on microcirculatory alterations in patients
with septic shock are independent of its systemic effects." Crit Care Med 34(2): 403-8.

De Backer, D., C. Verdant, et al. (2006). "Effects of drotrecogin alfa activated on

microcirculatory alterations in patients with severe sepsis." Crit Care Med 34(7): 1918-24.

Sakr, Y., M. J. Dubois, et al. (2004). "Persistent microcirculatory alterations are associated with
organ failure and death in patients with septic shock." Crit Care Med 32(9): 1825-31.

Spronk, P. E., C. Ince, et al. (2002). "Nitroglycerin in septic shock after intravascular volume
resuscitation." Lancet 360(9343): 1395-6.

Trzeciak, S., R. P. Dellinger, et al. (2007). "Early microcirculatory perfusion derangements in patients with
severe sepsis and septic shock: relationship to hemodynamics, oxygen transport, and survival." Ann Emerg
Med 49(1): 88-98, 98 e1-2.

20 Microscan Manual
 Quick Start Guide

1. Bring the device Quick Start and

to the bedside Guide
position the cart so the
laptop is easily viewed.
2. Hook up Microscan device.
3. Turn on microscan battery unit, be sure it is charged and not
plugged in.
4. Turn on computer, turn on.
5. Place cap on tip of the probe.
6. Open “Microscan by Studymaker” program.
7. Have the patient dry out the secretions in their mouth with
some gauze.
8. Input Patient Id, Operator, and Time point.
9. Obtain 5 videos of about 1 minute each from different
locations:
a.Choose good positioning.
b.Focus and adjust light intensity.
c.Avoid pressure artifact.
i. Find vessels, pull back until contact is lost, then slowly
advance.
ii.Use large venules as indicators – need good flow in the
venules.
iii.Find pressure with “fastest flow”.
10.Press record or C to start capture.
11.Images will be saved to the hard drive.
12.Upload as directed.
13.Shut down system and clean up the workstation.

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