User Manual
Contents
1. Biorep Technologies ISLET CELL COUNTER 3 ICC3 USER MANUAL Page 1 of 17 Biorep Technologies TABLE OF CONTENTS IN Ge aires sis sccce wkc eden snc esecdee doce wed sicedecdoetec ode eaddednces vacdesendeadaneasnudetecicetacus ences 3 1 1 SYMBOLS USED IN THIS VIA INA esse se ceases edncsa susdeneoaacandeaubaassanebeuses Susdenacastuadseculaqasanebeadeasnoeenes 3 Be INVENT INFOPMATION coreeana E E N 3 1 3 CORTA TE TFTORMA T o E ER 3 1 4 SAFETY INFORMATION ccceccsceccsceccsceccececcecscececsscscsscsceccecsceecsseecsceececescscnsescesescesescssescssescssesecess 4 1 5 PEA UII BSUS Wr cages tg gcse see cise sce Ge esses se deeds de as aeamacon te saneneiedea senda veccoase neces 4 2 INFRODUCTION ocrck AAAS 5 3 MACHINE FEATURES IDENTIFICATION ccccccccccccccccccccccccccccccccccccccccccccccccccccecs 6 TO ACCESSORI ee a eo A ee We eA ee eee ee 7 4 1 ISLET CELL COUNTER DISH ICC DISH ccc cceccccccscccsecceeccescceseceeuceescsueceeeceesceuseseeseusess 7 a OO FG rae ccc cases erences a a Ra a aa 7 5 1 TONIC TIONS recesses ccs ces doen erste E ats AEAN ETEEN A A 7 5 2 ISLET SAMPLE PREPARA TION ssssezicacsidecanediadvantaanidune baad sened ioesanedindvantbicitunebiad nened ibesanedindtactauciiusebiaceunt J 5 3 SAP STA N ereraa sce ts cones te EET AA EON 9 C COINCIDE aS 10 6 1 POWFR UP recesses rened NEETER 10 6 2 EEE 1i EEEE AE OE EAEE OOA A ASE A AE TOAS AA A EEE 10 6 3 SANPLE PREPARA TION zani2. does not allow adjustments to be made by the user This is actually a good thing The vision system is carefully calibrated at the factory to be in focus to have a consistent illumination and to have a pre set magnification factor It is this careful calibration that allows automated counts to be repeatable and comparable across machines and across research centers Page 6 of 17 Biorep Technologies 4 Accessories Some of these items must be ordered separately For ordering information please consult our webpage www biorep com or contact one of our sales representatives at 305 687 8074 4 1 Islet Cell Counter Dish ICC DISH The ICC Dish was specifically designed for use with the ICC vision system The central well is designed to provide enough area to allow the sample to be spread out to minimize errors due to aggregation but it is compact enough to allow for high resolution imaging of the region of interest The well is deep enough to minimize the risk of spillage during sampling and staining The dish is accurately positioned with a matching nest geometry on the ICC tray 5 Set up of the ICC 5 1 ICC Connections The ICC only requires 2 cables Power and communications The external power supply included is connected to the standard 2mm round DC power connector The power supply can be connected to a 110 or 220V outlet outlet adapter not included Establish a connection between the laptop and the vision
3. system through the network cable included 5 2 Islet Sample Preparation The recommended islet sample volume is 100 uL this is the default value pre filled on the sample information section For best results follow these best practices If counting stained preparations add 250 uL of DTZ solution See recommended DTZ solution preparation in the next section If counting an unstained preparation add 250 uL of buffer solution Page 7 of 17 Biorep Technologies The bottom of the dish must be completely covered in fluid Partial fluid coverage creates shadows at droplet boundaries that cause artifacts that affect detection and count performance The background of the image should be as a uniform as possible A fill volume between 350 and 400 uL is recommended to ensure the dish surface is completely covered Some slight leveling of the ICC dish might be required to distribute the fluid uniformly Do not overfill the dish Overfilling above 500 uL may have adverse effects on the illumination and the count accuracy Avoid air bubbles in the dish Large air bubbles cause similar problems to those of partial fluid coverage Small bubbles might cause localized errors and may even result in false positives The cell mass should be spread out as evenly as possible in the dish but away from the edges Agglomeration makes software segmentation harder and the results less reliable Cells close to the edges may be omitted since a small ma
4. 0 uL of islet suspension into ICC Dish 2 Pipette 250 uL of DTZ in the ICC Dish 3 Carefully level or gently agitate ICC dish to achieve an even distribution of the sample and uniform fluid coverage 4 Make sure there are no bubbles and that tissue is as evenly spread as possible 5 Place the ICC Dish in the Vision System B io re p Select stained or unstained and follow instructions Back J f Next Technologies The Sample Preparation screen includes a step by step guide to prepare the sample for counting The instructions change slightly depending if your sample is stained or unstained The default mode is stained Uncheck the box to count unstained preparations The GUI provides a live video feed of the sample so you can check the cell distribution within the ROI Remember that you want to spread the tissue as much as possible within the boundaries of the ROI only the cells within it will be counted Press Selecting on the loupe icon above the image will enable zooming Zoom in by clicking on the region you want to explore You can zoom out with SHIFT click If you wish to return to full screen view press the CENTER button Press Next to continue to the Sample Info page Page 11 of 17 Biorep Technologies 6 4 Sample Info S Islet Counter 3 3 9 0 em So Sample Info a ieee Donor Organ Operator Donor Unos Species Organ ID Enzyme I
5. D SAMPLE VOLUME uL 12345 Human m 12345 12345 100 Height m Donor Gender Organ Weight g User Account TOTAL VOLUME mL 16 Male 1 2 TEST 100 BMI Ethnicity Preservation Solution Date Dilution Factor 1 0 Hispanic No Selection x 10 24 2013 1000 Age yrs ABO Blood Group Time 20 A ial 12 21 PM Isolation Stage Layer Layer 1 Cause of Death Cold Ischemia Time Experiment T ml erimen e NA 0 hrs 0 min E Research Notes Write notes here a Enter General Info for the Experiment Back Next On the Sample Info page you can enter the information related to the sample being counted Donor information organ information can be filled with the keyboard or by selecting one of the choices in the drop down menus The Operator information is automatically filled The last column of fields on the right pertain to the Dilution Factor It is very important that you check that the dilution factor is correct before proceeding The machine has a default setting of 1000 100uL out of 100ml The Skip Analysis button on the top right is used when counting samples that have been stained for more than 10 minutes If your sample has been recently stained press Next to enter the Sample Analysis page Page 12 of 17 Biorep Technologies 6 5 Sample Analysis Islet Counter 3 3 9 0 Sample Analysis SKIP Analysis Center Save Img Lighting Staining Analyzi
6. d with all the related information see section X X The count results displayed are the following e DILUTION FACTOR is displayed as calculated from the information fields on the Sample Info page e OF PARTICLES displays the approximate number of individual islets present in the dish without taking their size into consideration e lEQ displays the total islet equivalent in the preparation as calculated from the sample contents and the dilution factor e AREA provides the islet area cover in millimeters squared This is thought to provide a very consistent metric of islet quantity which is not subject to size weightings like IEQ and is less sensitive to cell aggregation e PURITY provides a ratio between the area covered by islets and the total area covered by tissue in the sample Only available in STAINED mode Page 14 of 17 Biorep Technologies e B factor One of the challenges of accurate counting is related to sampling variability It is known that the predominance of large islets in a preparation can lead to significant variability of counts within the same preparation The presence or absence of a single large gt 400um diameter islet cell can vary the IEQ count of a 100 uL sample by a significant amount The B factor gives the user an idea of the impact of islets size distribution in the IEQ result A B factor of 1 means that there is no significant impact of size distribution As the B factor gets increases above 1 it si
7. en ETE 11 6 4 e LIN AET ETE E EE T E AAEE EEE EEEN E E EO EAE 12 6 5 AEE IN YS a E AR 13 The main function of the Sample Analysis page is to check the sample is properly prepared and that the uptake of DTZ by the islets is stable Start the Analysis by clicking the Analyze Image button A pop up will appear letting you know the time elapsed and allowing you to cancel the analysis at any time Analysis can be skipped at the user s discretion with the SKIP Analysis button on the top VU a EE AEAEE P E AEE A EAE AE O O EEE E EAE E E DE A E EE vores 13 The application looks for changes in the image that signal that staining is taking place Whenever this changes become stable the pop up will disappear and the LED indicators Lighting and Staining will turn Green allowing the user to continue to the Counting PAGE cccccccccccscerseeeseeeeeseeeececeeeeeeeeaaaaaaeeeeeees 13 CO COT a E A ee ee E E EES 14 C R OT a E E E E 15 7 CUSTOMER SERVICE sisvcssenssscissecereeuiucusinvaveiececesansisweceusvecevensicsneniadevsavsiisiuaisuesisiucisussaisdieaierseevatens 17 Page 2 of 17 Biorep Technologies 1 Getting Started WARNING Please read this section before unpacking installing or operating this machine Symbols Used in this Manual 1 1 The lightning flash with arrowhead symbol within an equilateral triangle is intended to alert the user to the presence of dangerous voltage within the machine s enclosure that
8. ethod is used every time An ICC in your lab will count with the same method as any other ICC out there making results more comparable across research centers and hopefully more significant Other software based cell counting technologies require training and rely significantly on user experience and input to perform accurate counts therefore they are also subject to user judgment and variability Furthermore these software packages are independent of imaging hardware such as microscopes illumination and cameras creating additional sources of variability The ICC is a self contained machine vision system that includes both hardware and software designed specifically for islet counting This ensures that magnification field of view illumination focus and other optical variables are constant making the measurements repeatable and reliable The ICC software is powerful yet its user interface is minimalist by design to facilitate an intuitive workflow Ease of use was a top priority when developing the ICC3 software which will guide the new user in a step by step wizard so that every islet sample is optimally quantified Page 5 of 17 Biorep Technologies 3 Machine Features Identification Power switch Power indicator LED Sample tray ICC Dish nest RJ45 connector to laptop Power supply connector oe SS YS Illumination Switch Not much right AS opposed to conventional microscopes the ICC Vision system
9. gnals that large particles are predominant If small islets are prevalent the B factor will be smaller than 1 It is up to the researcher to use and determine any acceptance criteria based on the B factor 6 7 Report For every count a full resolution image of the sample and a comprehensive report are saved in the ICC3 file folder on the desktop The report contains all the information results and relevant count statistics of the count as well as some graphical presentations of the IEQ results Date 10 3 2013 Time 4 15 AM UserID sore p Semple Information No Selection Sample Volume pl 100 00 Total Volume mL 100 00 Dilution Factor 1000 00 klet Equivalent IEQ 128635 429 Particle Count IPN 122 klet Area Cover I amp C mr 172 01 Purity 3 22 0 01 Sie Index B fector 13 Page 15 of 17 m Biorep Technologies The1 page of the report contains the time and date of the count and all the data related to the user and sample It also contains the numerical values of the results including Dilution factor IEQ Particle count Area cover Purity and B factor The 2 page displays a snapshot of the sample The 3 page contains graphical visualizations of the results The first graph represented by a bar graph shows the numerical quantity of particles in each IEQ size group Generally speaking the quantity is inversely proportional to the size group The smaller the islets the more the
10. may be of sufficient magnitude to constitute a risk of electric shock The exclamation point within an equilateral triangle is intended to alert the user to the presence of important information in the literature that accompanies the device 1 2 Machine Information In the spaces provided below record the Model and Serial No located on the bottom of your machine Model No Serial No RETAIN THIS INFORMATION FOR FUTURE REFERENCE 1 3 Contact Information Biorep Technologies Inc 3225 NW 112 Street Miami FL 33167 info biorep com www biorep com Tel 305 687 8074 Fax 305 688 8029 Page 3 of 17 Biorep Technologies 1 4 Safety Information WARNING DO NOT SKIP READING THIS SECTION PLEASE READ AND OBSERVE ALL WARNINGS AND INSTRUCTIONS GIVEN IN THIS USER S MANUAL AND THOSE MARKED ON THE UNIT RETAIN THIS BOOKLET FOR FUTURE REFERENCE DO NOT REMOVE THE MACHINE S COVER OR YOU MAY BE EXPOSED TO DANGEROUS VOLTAGE REFER SERVICING TO QUALIFIED PERSONNEL ONLY TO REDUCE THE RISK OF FIRE OR ELECTRIC SHOCK DO NOT EXPOSE THIS DEVICE TO WATER OR MOISTURE gt gt gt READ AND FOLLOW THESE INSTRUCTIONS 1 Keep these instructions for future reference and heed all warnings stated in this manual 2 Protect the power cord the power entry module and the plug from being walked on or pinched to avoid damage 3 Refer all servicing of the machine to qualified personnel Servicing is required when the apparatu
11. ng Image 3 00 08 Cancel Analyze Image y Press Analyze Image to continue Back iBiorep yze Imag __ Back a a Tech lanii The main function of the Sample Analysis page is to check the sample is properly prepared and that the uptake of DTZ by the islets is stable Start the Analysis by clicking the Analyze Image button A pop up will appear letting you know the time elapsed and allowing you to cancel the analysis at any time Analysis can be skipped at the user s discretion with the SKIP Analysis button on the top right The application looks for changes in the image that signal that staining is taking place Whenever this changes become stable the pop up will disappear and the LED indicators Lighting and Staining will turn Green allowing the user to continue to the Counting page Page 13 of 17 Biorep Technologies 6 6 Counting g Islet Counter 3 3 9 0 Counting Dilution Factor 1000 Center Save Img of Particles 163 IEQ IEQ dev 91035 24 Area mm 2 Area dev 1 12 0 9 Purity Purity dev 75 2 0 8 B Factor 14 New Sample Beta IEQ Results bIEQ plEQ Area IEQ 84117 71104 63655 oa Biore p Count Complete Technologies At this point the sample is ready to be counted Press Count Islets to generate a count When the count is complete a pop up will display the path where an automatic report has been save
12. nitors the stain uptake and will notify the user when the staining is stable Page 9 of 17 Biorep Technologies 6 Using the ICC 6 1 Power Up The main power is the green and only button on the front panel of the machine Connect the power cord provided in the accessory box to the module plug it into a 115 VAC or 230 VAC outlet and push the button A green LED indicator should turn on Make sure the network cable is connected to both the laptop and the vision system Power up the ICC laptop Boot into windows and double click on the ICC icon on the desktop 6 2 Login 3 Islet Counter v3 3 3 User Accounts Bior y Login Add User Delete User Edit User J Password A WARNING Password is case sensitive 2 Islet Counter v3 3 3 Version Press Login to continue The ICC application starts with a Log in screen Here you will be able to log in to your user account or create one by clicking on Add User Select your User Account from the drop down enter your password and click on Login to continue to the Sample Preparation screen The software application is closed from this screen by clicking on the red button The window close X has been disabled to prevent conflicts with the Vision System Page 10 of 17 Biorep Technologies 6 3 Sample Preparation z I islet Counter 3 3 9 0 Sample Preparation RES Center Save Img 1 Pipette 10
13. re are The second chart represented by a pie chart shows how much of the total IEQ is contributed by each IEQ size group With this representation it can be seen that despite the big difference in numerical particle quantities the contribution of each size group to IEQ is generally more uniform These graphical representations may allow the researchers to qualitatively assess the sample visually and with experience identify trends and patterns for different types of donors organs or isolation methods 3 G G 20 100 ICH 130 121 300 J01 250 6250 5000 101 3530 151400 300 Cell Group Relatie Contribution to IEC 5 Bsc 100 18c Miod 25 008 B131 2004e0 B2ct 230 8 gt 6252 30008 Eeee 33 2008 Peira Page 16 of 17 Biorep Technologies 7 Customer Service lf you encounter any problems please contact customer support at Biorep Technologies Inc 3225 NW 112 Street Miami FL 33167 info biorep com www biorep com Tel 305 687 8074 Fax 305 688 8029 Page 17 of 17
14. rgin close to the edge of the dish is excluded to minimize the risk of artifact Gently shaking the dish side to side in one direction helps spread the cells on the dish Swirling the dish in a small circle usually has the opposite effect and will tend to agglomerate the cells in the middle of the dish Use these two techniques to achieve a good spread within the region of interest ROI Page 8 of 17 Biorep Technologies 5 3 Sample Staining Staining the sample with dithizone DTZ drastically improves the count accuracy by enhancing contrast between acinar tissue and islets As with any other parameter concerning machine vision repeatability is the key factor Staining with DTZ has to be done in a repeatable and controlled fashion For best results the DTZ formulation composition the amount of DIZ added to the dish and the uptake time allowed for the islets to pick up the stain need to be consistent We recommend the following best practices Dithizone DTZ Solution Recipe a Dissolve 100 mg Dithizone in 20 ml Dimethyl sulfoxide DMSO and mix b Add 30 ml HBSS c Filter using a 0 2 or 0 4 um bottle top filter d Add 50 ml HBSS e Label appropriately and use on the day of preparation f Store at room temperature 20 28 C Uptake time should be at least 8 minutes before performing a count The value of the count may change as the cells get stained The Sample Analysis section of the software see section 6 5 mo
15. s has been damaged in any way 1 5 Packing List The following items are included with the purchase of an Islet Counter LFS123000D A8B Power Supply ICC3 UM ICC DISH Islet Counter Dish pack 10 Table 1 Packing List Page 4 of 17 Biorep Technologies 2 introduction This User Manual is intended to be used by scientists researchers and technicians who have already been trained in islet isolation procedures and technologies This document contains the necessary information for installing and operating the islet counter vision system and its software The Islet Cell Counter ICC has been developed to assist in the quantification of islet samples taken from isolated preparations The islet counter is a useful tool to determine yield in islet isolations or to quantify islet samples used for Perifusion experiments The islet counter was created to address the two main limitations of human counting Speed and variability Leveraging on machine vision technologies the ICC can perform counts and generate a report in minutes including IEQ area cover and purity Trained human counters require several minutes to perform accurate counts and calculate and fill the required metrics for documentation In addition it is known that despite standardization efforts islet counts can vary dramatically from one human counter to the other The adoption of machine vision technologies reduces variability and ensures that the same counting m
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