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Instrument Operating Manual

CyFlow® Cube 8 Rev-27_2015-07-02

Sysmex Partec GmbH

Am Flugplatz 13, 02828 Görlitz, Germany · Phone +49 3581 8746-0 · Fax +49 3581 8746-70 · [email protected] ·

www.sysmex-partec.com

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Table of content

Table of content ................................................................................................................ 3

Introduction ................................................................................................................ 5

Presentation ............................................................................................................... 6

2.1

Basics

..................................................................................................................

What is the Sysmex Partec CyFlow

Cube 8? ............................................................ 6

What are the applications for which the CyFlow

Cube 8 can be used? ..................... 6

What are topics covered by this manual? ................................................................... 6

What other manuals are available? ............................................................................ 6

What should I know before operating the CyFlow

Cube 8? ....................................... 6

2.2

In flow cytometry, what is..

. .................................................................................. 7

… a parameter? ......................................................................................................... 7

… a one-parameter histogram? .................................................................................. 7

… a histogram channel? ............................................................................................ 7

… the count in a histogram?....................................................................................... 7

… a peak? .......................................................................................................... 7

… background in a histogram? ................................................................................... 7

Instrument starting procedure

.....................................................................................

3.1

Sheath fluid level control and/or refill ................................................... 10

3.2

Switching on the CyFlow

Cube 8 ........................................................ 10

3.3

User levels ........................................................................................... 12

CyView

Controls

......................................................................................................

4.1

CyView™ Instrument Setting Controls ................................................. 14

4.1.1

1D/2D PLOTS

– display options ........................................................... 14

4.1.2

RESULTS ............................................................................................ 15

4.1.3

REGIONS ............................................................................................ 16

4.1.4

GENERAL - properties of the instrument ............................................. 17

4.1.5

ACQUISITION

– instrument control ..................................................... 17

4.1.6

MEASURE

– definition of measure modes........................................... 18

4.1.7

Measure modes ................................................................................... 19

CyView

Instrument/Measurement Settings ....................................................... 22

5.1

Priming / Initialization process .............................................................. 22

5.2

Work process ....................................................................................... 24

5.3

Shut down process .............................................................................. 24

5.4

Intermediate cleaning process ............................................................. 25

5.5

Sheath and Waste bottle ...................................................................... 25

Measurement parameters

...........................................................................................

Summary of Contents for CyFlow Cube 8

Page 1: ...Am Flugplatz 13 02828 Görlitz Germany Phone 49 3581 8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com CyFlow Cube 8 Instrument Operating Manual Revision Rev 27_2015 07 02 Issued by Sysmex Partec GmbH ...

Page 2: ...02828 Görlitz Germany Phone 49 3581 8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com 2 43 Imprint Copyright 2014 by Sysmex Partec GmbH Am Flugplatz 13 02828 Görlitz Germany Phone 49 0 3581 8746 0 Fax 49 0 3581 8746 70 Mail support sysmex partec com Web www sysmex partec com ...

Page 3: ...ter 7 a one parameter histogram 7 a histogram channel 7 the count in a histogram 7 a peak 7 background in a histogram 7 3 Instrument starting procedure 10 3 1 Sheath fluid level control and or refill 10 3 2 Switching on the CyFlow Cube 8 10 3 3 User levels 12 4 CyViewTM Controls 13 4 1 CyView Instrument Setting Controls 14 4 1 1 1D 2D PLOTS display options 14 4 1 2 RESULTS 15 4 1 3 REGIONS 16 4 1 ...

Page 4: ...perties 26 6 3 1D 2D Plot properties 27 6 4 Region ROI properties 28 6 5 Compensation 31 7 Typical Sample Analysis 32 8 Keyboard Mouse combinations 34 9 Appendix 35 9 1 Biohazards 35 9 2 Maintenance 36 9 2 1 Service 36 9 2 2 Transport and Storage 36 9 2 3 Disposal 37 9 3 Laser Safety 38 9 4 Technical Specifications 39 9 4 1 Example for Optical Standard Setup 39 9 4 2 CyFlow Cube 8 System 40 9 4 3 ...

Page 5: ...uously working on CyView to better fulfill your demands If you have questions concerning this manual or the software find problems associated with CyView or you have a good suggestion to be included in a new version please let us know by sending an email or a note to Sysmex Partec GmbH The present manual is valid as of CyView software version 1 5 For more details about the reagent kits suitable fo...

Page 6: ...tion for routine use and flexibility for research use for practically any flow cytometric application The applications cover Routine multi color immuno phenotyping Blood Cell Analysis HIV monitoring e g CD4 cell count Leukocyte Counting Rare Event Analysis Microorganism Analysis Fermentation Control Particle Concentration Analysis True Volumetric Absolute Counting Particle Size and Fluorescence Di...

Page 7: ...classes or channels In a one parameter histogram the channels are represented on the x axis the count in a histogram The number of cells being assigned to a given channel is referred to as channel content or simply count In a one parameter histogram the count is shown on the y axis a peak All cells having about equal characteristics among the analysed cell property e g content of a specific consti...

Page 8: ...tructure Forward Scatter FCS or Side Scatter SSC resp or on their relative fluorescence intensities in different light colors fluorescence parameters FL1 to FL6 In this example the dimension represented is the relative size FSC on a logarithmic scale in X and the number of events on a linear scale in Y Two peaks are visible Example of a dot plot A 2D dot plot presents correlated data over 2 dimens...

Page 9: ...t of immunological staining This histogram represents the spectrum of the cells presented in the previous dot plot stained with antibodies anti CD45 conjugated to Phycoerythrin PE Dy647 The X axis displays the fluorescence in a 4 dec logarithmic scale and the Y axis displays the number of events in a linear scale This dot plot presents the cells fluorescence in X on a logarithmic scale CD45 PE Dy6...

Page 10: ...at least once a week or before any daily use When filling up the sheath fluid bottle make sure no air bubbles are trapped in the yellow filter unit inside the bottle Make sure that the WASTE bottle is empty and the screw top is tightly closed The waste bottle must be emptied after and before each user session When using bio hazardous samples a volume of 50 ml of hypochlorite 0 5 Order No 04 4012 s...

Page 11: ...ER The code will be given to you by the main user s of the instrument The main user has the rights to define or delete users Main CyView login window The log in window allows to start the software at the USER MAIN USER or SERVICE level During start of the instrument the automated self testing procedures are processed Cleaning up and XML configuration displays the correct loading of the setting fil...

Page 12: ...tivate Main user followed by Build new account To create a new USER account type in name and password followed by Build new account Main user should be deactivated As User the own password can be changed by selecting the name and placing a new password followed by Change my password As Main user the own password can be changed by selecting the name and placing a new password followed by Change my ...

Page 13: ...8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com 13 43 4 CyViewTM Controls CyView Main Page after successful log in The main window will be your interface to acquire save re load and analyse your data Instrument real time display of workload L on board memory status M analysis volume V and analysis duration D ...

Page 14: ...As example 12Pl 6 Histograms 6 dot plots Plots are named H1 Hx for histograms and P1 Px for dotplots A specific plot is selected with the arrow keys Define a Comment characterizing the plot e g FSC Select X axis channel and Y axis channel in dot plots or X axis channel only for histograms Switch between Lin and Log scale for X and Y axis and change the Z axis scaling Define an Erosion level for do...

Page 15: ...ual regions according to the specified formula NumReg1 x NumReg2 x Scale DenomReg1 x DenomReg2 NumRegion1 2 defines numerators DenomRegion1 2 defines denominators NumOperator defines operator between 2 numerators x or DenomOperator defines operator between 2 denominators x or Unit allows to add text to the result table Scale introduces a factor to the formula Counter results on refers to the resul...

Page 16: ... plot the region refers to Color RGB defines the region s color and its color in color gating Max Count defines a maximum count for an Cells in Region particle limit see also Pages 17 19 Sorter Region On activates region as sorter region only in CyFlow Cube Sorter Color Gating On activates deactivates the region Use Delete and New to delete and create new regions Use arrow keys to switch between r...

Page 17: ...efines a factor for sample Dilution Clinic Customer display specific user information Defines sample port electrode Volume in µl µlPerSec mBar defines a sheath fluid flow parameter SW Version specifies current software version Rescale LSB shows area of automatic scaling Storemode defines FCS file format 4 1 5 ACQUISITION instrument control Select the trigger parameter multiple trigger parameters a...

Page 18: ...ed to define a fixed sample volume of 200µl Cells in region a particle number can be pre selected Speed values and Volume values can be edited and will be stored upon saving a configuration file Autoflags allows activation of specific functions Autostart automatic start of the measurement Activate the AutoStart flag Press start only once to confirm the process Connect a sample tube Autostart With ...

Page 19: ...aning cycle Subsequently the data can be stored In the measure modes VC electrodes and Analyze all the cleaning cycle will occur automatically as soon as the sample is finished No cleaning cycle will be started if the measurement is terminated by the end button Beware for all measure modes the sample analysis automatically stops when the sample is empty the stop electrode is reached even if the se...

Page 20: ...selected volume will be analyzed The volume can be used as the basis for concentration determination Volumetric Counting with Electrodes This measuring mode uses the START and STOP electrodes of the sample port to define a fixed sample volume In a standard sample port this counting volume is 200µl In a pre counting phase the sample is acquired normally as in continuous acquisition mode Reaching th...

Page 21: ... Am Flugplatz 13 02828 Görlitz Germany Phone 49 3581 8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com 21 43 Schematic overview of the measure modes with a flash indicates clearing of the data User selectable criteria in green software defined values in red ...

Page 22: ... loaded the PRIME mode is always selected User defined configuration files can start in PRIME or WORK mode depend on the status during saving In the following cases the PRIME mode should be performed Starting the instrument for the first time Daily initialization After the sheath fluid bottle was filled As trouble shooting procedure no bad signals blocking etc To start the priming initialization p...

Page 23: ...ing process automatically and guide through the program Connect a sample tube with Decontamination Solution violet solution Order No 04 4010 press Continue and wait until the system has finished Remove the sample tube press Continue and wait until the system has finished Connect the sample tube again press Continue and wait until the system has finished At the end press Continue The instrument wil...

Page 24: ...e port a separate cleaning mode is available The detail procedure would be the following Select the CLEAN batch by clicking on the CLEAN batch symbol After pressing Start the system will start the priming process automatically and guide through the program Connect a sample tube with cleaning solution green solution Order No 04 4009 and press Continue Connect a sample tube with decontamination solu...

Page 25: ...ion and reduce the background For this procedure connect a sample tube with cleaning solution and clean the instrument by clicking on the Clean symbol The instrument will perform one cleaning cycle Repeat the procedure with a sample tube filled with sheath fluid Please notice the differences between the clean modes If you choose the Clean symbol display at right lower corner the system performs on...

Page 26: ...e g only intact mammalian cells will be acquired Cell debris and smaller particles will be excluded as long as their FSC signal remains below the trigger threshold Multiple trigger The option allows to use multiple trigger parameters 6 2 Instrument setting properties The GAIN G and THRESHOLD T sliders must be adjusted to obtain the optimal gain maximum signal and minimum background Typically the F...

Page 27: ...me A default plot name Hx Px is defined in the file Default names of the parameters will be displayed The user can modify for each plot the parameter name line comment enter a name better matching the experiment 6 3 2 X Log On Y Log On This option will allow the user to change the plot s scaling Note that changing the scale will require you to adapt the gains of the PMTs 6 3 3 Erosion levels The e...

Page 28: ...ton To close the gate surface click mouse right 6 4 2 Region properties To access the region property Alt right click on the gate to edited On the PLOTS register on the right of the screen a selection of region options is displayed Region name As default the gate will be named region nx successive region region nx 1 The user can also define a name Remember to validate by Accept before to move to a...

Page 29: ...ogram splitter To create a vertical or horizontal histogram splitter please move the cursor into the histogram and press the right mouse button Select Built vertical splitter region group Built horizontal splitter region group to divide the histogram into two sections VS1 1 and VS1 2 The intersection can be modified by moving the cursor into the plot and keeping the left mouse button pressed 6 4 5...

Page 30: ... color with Color RGB To activate changes press Accept 6 4 7 Applying regions to other plots Gating Function Clicking into any histogram or plot with the right mouse button outside a region area allows to select any existing region Reg1 Regx which is labeled with Color gating on Display options are Show all events shows all events in pseudo 3D color Show color regions only shows the events in the ...

Page 31: ...581 8746 70 info sysmex partec com www sysmex partec com 31 43 6 5 Compensation Press Compensation button to open the compensation Compensation Select a parameter combination to compensate Use the slide bar to set the compensation Clear Save and Load Compensation Confirm by clicking Accept Example of a color crosstalk compensation of FL1 into FL2 ...

Page 32: ...e load Perform PRIME process Fill a sample tube with 1 2 ml of your sample Check the tube for eventual imperfection or contaminants cracks aggregates hair and replace or remove resp if necessary Insert the tube into the sample port push it up until a distinct click is heard Press the start button In the console window information on the status of the measurement is displayed The following work is ...

Page 33: ...lations of the instruments set up or Space at the keyboard By default the continuous measure mode is pre selected In this case the measurement will only stop automatically when the sample is consumed An earlier stop can be realized by pressing the STOP button Data can be saved with The configuration file can be saved with Besides the Analyze all measure mode other measure modes can be selected Vol...

Page 34: ...d left mouse Moves a region in standard steps Ctrl Moves a region in small steps click and hold left mouse Region Shift Moves a region to the original position click and hold left mouse Shift Ctrl Moves the region by addition of an interval to the original position in very small steps click and hold left mouse click and hold left mouse Move the regions name over 5 positions N E S W central M Right...

Page 35: ... partec com 35 43 9 Appendix 9 1 Biohazards Please note Strict guidelines international as well as national regulatory standards such as PPE personal protection equipment must be met for all users Therefore the system is marked with the following biohazard label Warning The Waste may contain biohazardous and carcinogenic material from the samples infectious material dyes Warning biohazards ...

Page 36: ... brush and flush with clean distilled water several times Remember a clean sheath fluid reservoir is critical for proper operation If the CyFlow Cube will not be used for longer periods clean flow system by using distilled water Put a sample tube half way filled with distilled water at the sample port Clean waste and sheath reservoir wipe top dry 9 2 1 Service All service is to be made from an aut...

Page 37: ...sposal In case of product disposal please proceed according to the Sysmex Partec standard operating procedure SOP for decontamination After decontamination the system has to be disposed according to the local regulations and laws For further information please contact your local distributor or Sysmex Partec Warning The Waste may contain biohazardous and carcinogenic material from the samples infec...

Page 38: ...product according to the EN 60825 1 2007 Please note Laser light can be emitted if the housing of the device is damaged and the protection cover for the laser beam is removed Therefore the system is marked with the following laser safety labels Warning It is prohibited to open the instrument as it is equipped with a class 3b laser unit Warning laser radiation Attention Laser radiation Class IIIb i...

Page 39: ...88 nm laser PE FL3 blue fluorescence fluorescence origin UV LED DAPI FL4 red fluorescence fluorescence origin 638 nm laser APC If required by a specific application the optical standard setup can be optimized by exchanging preassembled removable mirror filter blocks This is a matter of seconds and does not require any re adjustment The CyFlow Cube flow cytometer can be equipped with various light ...

Page 40: ...sis True volumetric absolute counts counting per volume True Volumetric Based on precise counting and mechanical fluid volume measurement Absolute Counting No need for reference sample or beads Instrument Check Sysmex Partec Count Check Beads No 05 4010 Sysmex Partec Calibration Beads 1µm No 05 4007 Sysmex Partec Calibration Beads 3µm No 05 40018 Sysmex Partec DNA Control PI No 05 7303 Sysmex Part...

Page 41: ...5 µm x 100 µm at 488 nm other beam geometries upon request 9 4 4 CyFlow Cube 8 Fluidics Fluidic System Completely closed system for sheath water and sample volumes no fluid droplets or aerosols generated nor released from the instrument Flow Cuvette Synthetic quartz flow cuvette 350x 200 µm for laminar sample transport with sheath fluid fluorescence forward and side scatter light detection Sample ...

Page 42: ...ment Operating Manual CyFlow Cube 8 Rev 27_2015 07 02 Sysmex Partec GmbH Am Flugplatz 13 02828 Görlitz Germany Phone 49 3581 8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com 42 43 Notes ...

Page 43: ...Sysmex Partec GmbH Am Flugplatz 13 02828 Görlitz Germany Phone 49 3581 8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com ...

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