TITLE: Non-invasive sublingual glycocalyx measurements using GlycoCheck (RRID:SCR_018254)
AUTHORS: Tracey Weissgerber, Oscar Garcia Valencia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes procedures for non-invasive measurements of sublingual vascular glycocalyx degradation using the GlycoCheck system (RRID:SCR_018254). This includes procedures for setting up the equipment, performing tests, saving data, shutting down the system, troubleshooting and removing dust from the camera lens.</div></div>

[STEPS]
?. [Set-up (to be completed before the patient is tested)]
Take out the computer and camera.
?. [Set-up (to be completed before the patient is tested)]
Plug camera into a USB port on the right-hand side of the computer
?. [Set-up (to be completed before the patient is tested)]
Turn on the computer
?. [Set-up (to be completed before the patient is tested)]
Select Glycocheck User
?. [Set-up (to be completed before the patient is tested)]
Wait until “Process Scheduler” status is shown as “Idle” on the taskbar
?. [Set-up (to be completed before the patient is tested)]
While you are waiting:a.     Take the storage probe cover off and place in the camera spot of the suitcaseb.     Open the end of the packaging for a new probe coverc.     Gently place the probe cover on the camera, leaving it in the packaging
?. [Set-up (to be completed before the patient is tested)]
Wait for “results: OK” message
?. [Set-up (to be completed before the patient is tested)]
Open Glycocheck-New program
?. [Set-up (to be completed before the patient is tested)]
Enter password
?. [Set-up (to be completed before the patient is tested)]
Wait for database synchronization to complete
?. [Set-up (to be completed before the patient is tested)]
On the calibration screen, select “Camera KK”
?. [Set-up (to be completed before the patient is tested)]
Click “Start Calibration”
?. [Set-up (to be completed before the patient is tested)]
Select “Add new patient” from the drop-down menua. Enter patient IDb. Under birthday, enter test datec.  Under “Last name” enter first 3 letters of the last named. Under “First name” enter first 3 letters of the first namee. Enter genderf. Click “Add patient” to add the patient to the database
?. [Set-up (to be completed before the patient is tested)]
Click “Admission” to ensure that the patient always appears in the list on the right
?. [Set-up (to be completed before the patient is tested)]
Click on the battery charging signal in the lower right corner of the computer screen. Ensure that “High Performance” is selected
?. [Set-up (to be completed before the patient is tested)]
Plug Hard Disk Drive into a USB port on the right-hand side of the computer
?. [Testing a participant]
Place a paper towel in front of the computer in case of drooling
?. [Testing a participant]
Give instructions
?. [Testing a participant]
Make sure the correct participant is selected in the “Admissions” List
?. [Testing a participant]
Ask participant to swallow to clear saliva (reduces air bubbles)
?. [Testing a participant]
Wait until participant places camera under tongue
?. [Testing a participant]
Press “Perform selected visit”
?. [Testing a participant]
If the participant needs a break, press “Abort”; when the participant is ready to resume testing, press “Cancel”
?. [Testing a participant]
Focus the camera once the subject finds a stable image; continue to focus throughout the test as needed, being careful not to move the camera
?. [Testing a participant]
If you see a crystalline structure, the camera is focused on the plastic cover and not on the tongue. Turn the black knob a lot until you see vessels; then keep turning until flowing red blood cells are in focus
?. [Testing a participant]
If the camera is very out of focus and video is being collected (green bar going up) despite the fact that no vessels are visible and in focus, wait until you have focused the camera; then stop the trial and start over. Do not use data from this trial.
?. [Testing a participant]
We need several videos of different vessels, obtained from slightly different locations. Once the green bar goes up by 1/5 to 1/6 in a particular location, as the participant to move the camera very slightly until a new set of vessels is visible; then hold while the camera records. Repeat this pattern of a stable period of recording followed by a small movement to find new vessels, until recording is completed. If the participant has a difficult time keeping the camera stable, this may not be needed
?. [Testing a participant]
If you only see a few very small, looping vessels, encourage the participant to move the camera towards the back and center of the tongue
?. [Testing a participant]
Remind the participant to breathe through his or her nose
?. [Testing a participant]
If there is an air bubble, or the red air bubble warning light is on, the camera will not collect video (no change in height of green bar). Ask the participant to move the camera slightly until the air bubble is no longer in the image.
?. [Testing a participant]
If cells are not flowing, are flowing slowly, or the direction of flow is changing, remind the participant to have the camera gently touching the tongue without pressing into the tongue.
?. [Testing a participant]
If the 5:00 timer runs out, click “continue” to give the participant more time to complete the trial. Note that extra time was needed in the trial comments (see instructions below).
?. [Testing a participant]
The program will stop automatically when data collection is complete. Take the camera and place the probe cover back in the protective sleeve.
?. [Testing a participant]
Add your comments in the field that opens once the trial is completeda. Time of dayb. Whether or not the patient is fasted; if not fasted, note time of last mealc. Whether or not the patient had caffeine in the six hours prior to the testd. Note if extra time was needede. If the data should not be used, state this and list the reason why the data should not be usedf. Note any other important factors that may affect the outcome of the test – stress, smoking, medications, position of the patient, etc.
?. [Testing a participant]
Click “Next patient” – the program will start calculations after 30 seconds. Two trials can be done back to back by clicking perform new visit before the 30 second mark.
?. [Testing a participant]
Go to “Patient Screen”. This button will take a minute to appear if this is the first trial.
?. [Testing a participant]
Wait for data to appear after calculations are complete (5 min). Do not unplug, shut down, remove camera or attempt to start a new trial while calculations are in progress.
?. [Testing a participant]
Once the data appear, click “Home”
?. [Testing a participant]
Perform 3 trials per participant by repeating steps 20-29
?. [Testing a participant]
Show picture of blood vessels underneath the tongue – we are looking for big vessels at the back of the tongue, just to the right or left of the center
?. [Testing a participant]
Raise tongue slightly, place the camera under tongue just touching the surface (not pressing, no pressure)
?. [Testing a participant]
The camera flashes a bright green flashing LED light when turned on. To avoid discomfort, we will ask you to place the camera under your tongue before we turn it on.
?. [Testing a participant]
Demonstrate how to hold the camera – with two hands wrapped around base, forearms or arms supported on desk
?. [Testing a participant]
Tell the participant that you will focus the camera by turning the black knob; you will talk to them throughout the protocol to give them instructions as needed
?. [Testing a participant]
The participant can use the paper towel if he or she starts drooling.
?. [Saving the data]
Go to “Patient Screen” to export summary data calculated by program
?. [Saving the data]
Go to “Measurement Screen” to export detailed data for each trial
?. [Saving the data]
Click “Export Data”
?. [Saving the data]
Save on USB
?. [Saving the data]
Save patient test summary (patientreport.csv)
?. [Saving the data]
Select the trial that you want to export
?. [Saving the data]
Click “Export data”
?. [Saving the data]
Save on USB
?. [Saving the data]
Adjust filename: add T1 to filename for trial 1, T2 to filename for trial 2, T3 to filename for trial 3
?. [Saving the data]
Repeat steps 31.1 to 31.4 until all trials are saved
?. [Shutting down the camera]
Never unplug, shut down or detach the camera until all calculations are complete. When all calculations are complete, close the Glycocheck program.
?. [Shutting down the camera]
Shut down the computer
?. [Shutting down the camera]
Detach the camera
?. [Shutting down the camera]
Remove and discard the used probe cover; replace the storage probe cover. Never leave the camera unprotected without a probe cover.
?. [Shutting down the camera]
Place the camera in the box, carefully coiling the cord so that it fits in the cord space in the suitcase
?. [Shutting down the camera]
Ensure that the computer battery is fully charged for the next trial
?. [Shutting down the camera]
Wipe the camera handle with a disinfecting wipe.
?. [Shutting down the camera]
Place the battery in the lower part of the suitcase
?. [Shutting down the camera]
Place the computer in the suitcase, with the tongue picture and patient schedule on top
?. [Shutting down the camera]
Ensure that the camera cord is not sticking out, and will not be damaged when closing the suitcase
?. [Troubleshooting]
Message “Error Ocurred Scheduler Service is not running”
?. [Troubleshooting]
If the Glycocheck program gives an error and won’t start or won’t complete measurements, restart the computer.
?. [Troubleshooting]
Right click on Process scheduler and select “Start Service”
?. [Troubleshooting]
If that does not work open task manager and on the “Services” tab look for “gmt-pss” and make sure is running. If it is not then right-click it and select start service.
?. [Troubleshooting]
If steps 42 and 43 don’t work, email Glycocheck
?. [Cleaning camera lens]
If you see dark specks on the image, indicating that there is dust on the lens, use the following procedures to clean the lens.
?. [Cleaning camera lens]
Remove the protective camera cover
?. [Cleaning camera lens]
Remove the camera extension (long, pointed portion of the camera that goes under the participant's tongue)
?. [Cleaning camera lens]
Replace the camera and put on a new lens cover
?. [Cleaning camera lens]
Insert a lint free cotton swab with a very long handle into the lens and rub gently to remove dust
?. [Cleaning camera lens]
Run a test to determine whether the lens is now free of dust and debris. If not, repeat steps 45.1 to 45.4 until the lens is clean. When finished, replace the protective lens cover.
?. [Troubleshooting]
Error message: Licsense is expired. Contact GlycoCheck and ask them to update the license.