microscan manual v1_05web

Gabriel Najarro, Nathan Shapiro, MD

Microcirculation capture by Microscan

2008-2010

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 SignalingThe 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 FlowThe 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, MPHDon Yealy, MD C. Ince, PhD Stephen W. Trzeciak, MD

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Table of ContentsBackground 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

Microscan Manual 3

Microcirculation capture by MicroscanMicrocirculation capture by Microscan

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.

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.

Microscan Manual 5


Microscan

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 Manual 7


Microscan Head1. Light Intensity2. Tip Ejector3. Video Connector4. Indicator LED5. Focus6. Removable Handle

Microscan Battery Unit1. Power2. Charger Connection3. Video Connector from

Microscan Head4. Video Connector to ADVC5. Power LED6. Charge LED

Extras1. Microscan Imaging Head2. Spatial Calibration Unit3. Removable Handle4. Battery Unit5. Power Plug6. AC Adapter7. Disposable Tips

Hardware

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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Analog to Digital Video Converter (ADVC) Video Output Power

Video Input Signal Selector

Front Back

Connection Diagram

Microscan Manual 9


Connecting the Microscan to the Computer1. Connect gray cable A to Microscan Head.2. Connect gray cable A to Microscan Battery

Unit.3. Connect black cable B to Microscan Battery

Unit “Video Out”.4. Connect black cable B to front ADVC “Video

In”.5. Connect 6 pin end of Cable C to back of

ADVC6. Plug in the appropriate power adapter into the

back of the ADVC7. Connect the 4 pin end of Cable C to the

Laptop8. Remember to activate the Signal Selector on

the ADVC once everything is connected and powered on

Cable A

Cable B

2 3

Cable C

6 pin end

4 pin end

4

Front

5

7

ADVC

Microscan Head

Battery Unit

Laptop

Back

6

1

Software - Capture

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The software we use to capture videos is called Microscan by Study Maker and is very straightforward to use.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 quality7. If the video contains excessive movement,

bubbles, cloudy saliva, blank space etc. You can delete the scan. *NOTE* this will permanently delete the scan so be careful

8. Use the comments section to make any notable characteristics about a particular scan.

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3

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5

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6

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Software - Upload Manager

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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. Enter your email address for identification purposes

4. Click Enter & Proceed

5.You should have a list of all the files pending upload

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

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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 curl their tongue towards the back of their mouth. Once 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.

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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 11. 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 bed8. Use your non-dominant hand to stabilize your dominant hand between holding the Microscan

device and the patient’s chin.

Microscan Manual 13


Bedside UsePositioning: Method 21. 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 bed4. 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 UseObtaining Images1. 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 stability4. 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.

Microscan Manual 15


Image QualityObtaining 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

Microscan Manual 17


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.

Image Quality

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

Looped vessels

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 ArticlesBateman, 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 ResearchDe 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 onmicrocirculatory 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 withorgan 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 volumeresuscitation." 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.

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Quick Start Guide

Microscan Manual 21

1. Bring the device to the bedside and 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.

Quick Start Guide

microscan manual v1_05web

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