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24

CyFlow

™

Space | Operating Manual | March 2021

9 Instrument

Settings

Instrument settings are used to optimize the CyFlow™ Space acquisition for the
particles of interest. The adjustments cover the gains of the optical detectors, e.g.
the photomultiplier high voltages, the amplification mode (lin, 3 or 4 decade
logarithmic), lower and upper level thresholds and sample speeds. Instrument settings can
be saved for a given application and re-loaded for later use.

The following pages will describe the instrument settings that can be made. Please also
refer to the other referenced software manuals for details on how to change the instrument
settings.

Figure 7: Multiparameter instrument settings box

9.1 The Parameter Setup Dialog Box

Opening the Parameter Setup

Before starting an acquisition, assure the parameters of interest are
selected and set up according to your analysis.

Click the "Parameters..." button in the Instrument Settings box.

The Parameter Setup Dialog Box appears.

Figure 8: The Parameter Setup Dialog Box

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Summary of Contents for CyFlow Space

Page 1: ...For Research Use Only Not for use in diagnostic procedures With FloMax 2 1x software Doc No CY S 3001R FM2IFUEN Rev 013 Rev date 18 03 2021 EN CN 391 CyFlow Space Operating Manual ...

Page 2: ...eal time Data Processing and Results 9 3 4 Absolute Cell Counting 10 4 Safety 11 4 1 Laser radiation hazards 11 4 2 Electrical hazards 11 4 3 Alterations to the device 11 5 Operating Basics 12 5 1 Switching on the CyFlow Space 12 5 1 1 Check SHEATH and WASTE bottle 12 5 1 2 Switch on the instrument 12 5 1 3 Control of the 488 nm laser output power for 200 mW high power laser only model Coherent Sa...

Page 3: ... Trigger 25 9 1 2 Gain 26 9 1 3 Sample Speed 27 9 1 4 Threshold Lower Level L L 29 10 Disposal 30 10 1 Disposal of device 30 10 2 Disposal of components 30 10 3 Waste disposal 30 10 4 Sheath Fluid disposal 30 11 Appendix 32 11 1 Installation and uninstallation 32 11 2 Installation Requirements 32 11 3 Instrument Setup Step by Step 34 11 4 The Flow Cuvette 37 11 5 Maintenance and Service 39 11 5 1 ...

Page 4: ...or photo multiplier 22 Figure 6 Optical geometry at the laser interrogation schematics 23 Figure 7 Multiparameter instrument settings box 24 Figure 8 The Parameter Setup Dialog Box 24 Figure 9 Sample Transporting System of the CyFlow Space 28 Figure 10 Recommended placement of CyFlow Space components 32 Figure 11 Instrument Setup and periphery CyFlow Space shown from the rear 33 Figure 12 Computer...

Page 5: ...tification 1 1 Product information Name CyFlow Space Software FloMax 2 1x REF CY S 3001R 1 2 Manufacturer Sysmex Partec GmbH Arndtstraße 11 a b 02826 Görlitz Germany Phone 49 3581 8746 0 Fax 49 3581 8746 70 info sysmex partec com www sysmex partec com ...

Page 6: ...alysis Cell Counting Rare Event Analysis Cell cycle and cell proliferation Analysis Detection and Analysis of Microorganisms and Viruses Analysis of Environmental Samples Fermentation Control Particle Size and Fluorescence Distribution Analysis 2 3 What topics are covered by this manual The CyFlow Space Instrument Operating Manual covers the basic operation and maintenance of the CyFlow Space inst...

Page 7: ...operating the device Emphasis lays on installation start up and safe operation of the device If you have any questions about the content of the Operating Manual or operating the device please contact your local Sysmex representative 2 7 1 Symbols in this document To make warnings more accessible the following symbols are used to reference the kind of hazard1 Hazard symbol Meaning General hazards B...

Page 8: ...ING Names the source of the hazard Describes the consequences of the hazard This could result in death or severe personal injury Specifies the steps that must be taken to eliminate the hazard CAUTION Names the source of the hazard Describes the consequences of the hazard This could result in minor injury Specifies the steps that must be taken to eliminate the hazard NOTICE Names the source of the ...

Page 9: ...e individually illuminated by the light spot of the laser Due to the excitation the dye molecules emit fluorescence of characteristic colour emission wavelength spectrum This fluorescence light is separated into colour ranges by means of optical filters The intensity of each colour range is analysed for each single cell Besides fluorescence the intensity of light scattered by each cell can be meas...

Page 10: ...in histogram and or dot plot diagrams 3 4 Absolute Cell Counting Since the CyFlow Space analyses all cells passing through the flow cell while precisely monitoring the fluid volume of the sample it allows volumetric counting during the analysis i e the determination of concentration of any cell subpopulation Cell subpopulations can even be defined and their concentration analysed at a later date a...

Page 11: ...xposure causes damage to skin and eyes Avoid direct exposure 4 2 Electrical hazards Please place the device in such a way that the wiring is easily accessible to unplug and does not suffer damages in any way Only replace removable wiring with adequate spare parts 4 3 Alterations to the device Unauthorized alterations to the device or the software result in risks towards all user groups and the dev...

Page 12: ...ton close by Also switch on the 488 nm laser please see chapter 10 2 Instrument Setup Step by Step Figure 13 638 640 nm lasers are software controlled or are switched by additional labelled hardware switches next to the 488 nm laser power switch e g the 405 nm the 375 nm and the 532 nm lasers 5 1 3 Control of the 488 nm laser output power for 200 mW high power laser only model Coherent Sapphire Th...

Page 13: ...re not to come in contact with the fluid In case of accidental contact wash your skin throughout with soap and disinfectant 5 2 Multi laser Measurements NOTICE Alignment of lasers must only be done by Sysmex representatives or by authorized service personnel The CyFlow Space Flow Cytometer can be equipped with one up to 4 laser light sources Actual optical setup of your instrument may therefore va...

Page 14: ...laser the leading laser first and then through the laser s at spot no 2 and no 3 if present Therefore all parameters derived from a laser located at a spot other than no 1 require a time delay Time delay of detectors is set in the Parameter Setup dialog box see chapter 9 1 5 3 Starting a Measurement NOTICE Please see FloMax Acquisition and Instrument control on how to change instrument settings Ma...

Page 15: ...cquisition finishes automatically To finish the acquisition before click the END button or simply remove the sample tube from the sample port In any case the instrument initiates now the REPOSITION phase to prepare the instrument for the next analysing cycle or A Biosafety cleaning cycle is started The system status changes to Clean and then Ready The system is ready for the next sample NOTICE Whe...

Page 16: ...ncubate for 15 minutes Re start the system by pressing START and let it run to the end Run the system with 1 6 ml Cleaning Solution until the sample tube is empty Run 1 6 ml Sheath Fluid for two minutes Press STOP 2 Protect the sample port Leave a test tube filled with sheath fluid connected to the sample port this avoids drying and crystallization of any remaining material in the tubing 3 Quit th...

Page 17: ...of unknown concentration Disadvantages Depends on the accuracy of the specified beads concentration Constant running costs for the reference beads 6 2 True Volumetric Absolute Counting To overcome the drawbacks of the Dual and Single Platform Technique with reference beads Sysmex Partec instruments additionally offer an alternative way of absolute cell counting which is based directly on the basic...

Page 18: ...he sample tube The analysed volume is exactly 200 µl counting volume Effects of the sample meniscus in the tube are eliminated because the START and STOP electrodes are arranged symmetrically The sample conductivity does not influence the volumetric measurement as long as the fluids can be detected electrically 6 3 Precision and Reproducibility Precision and reproducibility of the Sysmex Partec in...

Page 19: ...on solution Significant sedimentation can take place within minutes The CyFlow Space analyses the concentration at the location of the sample uptake where it can vary over time due to sedimentation or de sedimentation effects Consequently avoid too long count phases due to low sample speed e g below 2 µl s Count Time typically should not exceed 2 4 minutes 1 Shake sample for re suspension of parti...

Page 20: ...ntrations To achieve numbers per ml multiply the count with the factor 5 count in 0 2 ml x 5 count ml If the function Clear Histograms on Count Start is disabled FloMax will label the data set acquired during COUNT phase but will maintain all data of RUN and COUNT phase In FloMax Cell concentration will be calculated on basis of the data label In 3rd party software a calculation of concentration i...

Page 21: ...erent purposes Exemplary standard configurations e g include 7 1 2 Laser instrument Instrument is equipped with a blue diode pumped solid state Laser 50 mW at 488 nm and a red laser diode at 638 nm 25 mW Up to eight optical parameters can be included colours reflect laser origin of the signal FSC forward scatter SSC side scatter FL1 green fluorescence FITC FL2 orange fluorescence PE FL3 red fluore...

Page 22: ... 3 Other parameter allocations possible The instrument configured as flow cytometer uses a second optical bench allocating 8 additional detectors extending the number of detectors to 16 Information on the optical configuration of a specific instrument is displayed in the optical layout scheme of an instrument which can be found in the individual instruments documentation The instrument is equipped...

Page 23: ...e flow cell Laser light scattered from the particles is detected in the forward direction range forward scatter Side scatter and fluorescence light is collected by one objective with cell sorting option or two objectives flow cytometer w o cell sorting option at a right angle sample hydrodynamic focusing laser beam side scatter fluorescence forward scatter sheath fluid waste A ...

Page 24: ...r a given application and re loaded for later use The following pages will describe the instrument settings that can be made Please also refer to the other referenced software manuals for details on how to change the instrument settings Figure 7 Multiparameter instrument settings box 9 1 The Parameter Setup Dialog Box Opening the Parameter Setup Before starting an acquisition assure the parameters...

Page 25: ...ams from different lasers are directed to different positions spots of the flow cuvette see chapter 3 2 Flow Cytometry Analysis Therefore all laser derived signals from a laser at a spot other than spot no 1 need to be analysed with a time delay of 50 µsec 9 1 1 Trigger Leading Trigger In order to discriminate particles of interest e g cells from other particles e g cell fragments or nutrition par...

Page 26: ...clude the red particles from the analysis triggering on red would exclude the green If the interest is in both subpopulations it is required to trigger on green and red simultaneously This can be done with Trigger All Trigger parameter s and mode can be selected in the instrument settings box Please see FloMax Acquisition and Instrument Control for details 9 1 2 Gain NOTICE Gain and lin log amplif...

Page 27: ...n by a factor of two Gain values between 250 and 600 are a good start for adjustment For each parameter you can choose between linear or logarithmic amplification In logarithmic amplification mode the signal range can be set to 3 log3 or 4 decades log4 corresponding to a range of 1 1000 or 0 1 1000 Generally a logarithmic amplification is advisable if particles with a broad range of intensities ab...

Page 28: ...ing Manual March 2021 Typical Speed Values a High accuracy measuremens e g DNA with 1 5 CV Speed 0 5 µl s b Fast measurements and absolute counting Speed 4 0 10 0 µl s Figure 9 Sample Transporting System of the CyFlow Space ...

Page 29: ...ettings Figure 7 Start with a low L L e g 10 Increase the L L until no more of the small noise signals appear in the histograms Make sure not to remove signals from particles of interest by a L L being too high A L L range of 0 999 9 corresponds to the full histogram scale independent of the channel resolution actually selected If a leading trigger is selected then the L L can only be effectively ...

Page 30: ...ypochlorite Solution Incubate the mix of waste and Hypochlorite Solution for about 15 minutes INFORMATION When screwing or unscrewing a bottle and its respective Cap it is advantageous to twist the bottle The Tubing must stay unbent 1 Unscrew the Waste bottle Beware of possible vacuum 2 Secure Cap and Tubing on a clean surface in a stable manner 3 Decontaminate the Bottle with Hypochlorite Solutio...

Page 31: ...ubing on a clean surface in a stable manner 3 Dispose of Sheath Fluid according to your local legislation and its associated labelling 4 Screw down the Cap by turning the Bottle Do not twist the Tubing and the Sensor cable Close the bottle tightly 5 Wipe bottle with clean cloth 6 Dispose of cloths ...

Page 32: ...y with the following criteria when positioning the device Place the device on a solid dry and clean horizontal surface Place the device and the Sheath Fluid and Waste bottles on surfaces of identical height e g the same table at a distance corresponding to the length of the sensor cables and tubing Avoid smoke dust vibrations direct sunlight and any source of direct heat Leave a minimum distance o...

Page 33: ... from the rear CyFlow rearside Screen Computer rearside Printer CCD Camera Interface Monitor Printer CCD Computer AC AC AC Air Sheath Mouse Keyboard Sheath Waste AC Waste CyFlow rearside Screen Computer rearside Printer CCD Camera Interface Monitor Printer CCD Computer AC AC AC Air Sheath Mouse Keyboard Sheath Waste AC Waste CMOS ...

Page 34: ...e the system 1 Setting up the Computer Connect printer screen keyboard and mouse to the computer with the appropriate connection cables Connect computer screen and printer to AC power line Figure 12 Computer connection on the CyFlow Space rear 2 Connecting the CyFlow Space to power line The CyFlow Space must be operated with AC 100 240 V 50 60 Hz ...

Page 35: ...instrument Connect the CyFlow Space to the computer with the interface cable The laser connection is only required for the 200 mW 488 nm laser There is a CMOS camera inside the CyFlow Space optical bench to monitor the focus of the objective and the sample flow inside the cuvette Connect the camera signal outlet CMOS to the Video In connection of the computer with the video connection cable Networ...

Page 36: ...ensors on the right side of the CyFlow Space Connect the waste fluid container to the waste outlet at the right side of the CyFlow Space All tubes are labeled accordingly NOTICE Make sure that the sheath and waste fluid bottles stand on the same level as the instrument to prevent that hydrostatic pressure is influencing the sheath fluid pressure ...

Page 37: ...waste connections WARNING This device is a class 1 laser product while the housing is unopened If the housing is opened and laser light is emitted the device is a class 3b laser product Laser light exposure causes damage to skin and eyes Avoid direct exposure Do not open the device without authorization by Sysmex Partec NOTICE Air bubbles usually enter the flow cuvette through the sheath fluid inl...

Page 38: ...e the disturbance As first easy approach to release bubbles during system run attach a tube with Cleaning Solution and pinch first the sheath and then the waste tubing several times to remove air bubbles through the flow channel into the waste In case air bubbles are still persistent please apply the following procedure 1 Select the instrument settings Sample pre run 15 sec Stabilizing time 5 sec ...

Page 39: ...voir is critical for proper operation If the CyFlow Space will not be used for longer periods clean flow system by using distilled water Put a sample tube half ways filled with distilled water at the sample port Clean waste and sheath reservoir wipe top dry 11 5 2 Service All service is to be made by authorised service personnel Please contact your local Sysmex representative 11 6 Transport and St...

Page 40: ...using is unopened If the housing is opened and laser light is emitted the device is a class 3b laser product Laser light exposure causes damage to skin and eyes Avoid direct exposure Do not open the device without authorization by Sysmex Partec Figure 16 Warning laser radiation Figure 17 Additional explanation Attention Laser radiation Class IIIb if cover is removed and shutter is opened Avoid eye...

Page 41: ...t should be avoided Applications Research applications Immunophenotyping DNA Analysis Apoptosis Blood Cell Analysis Leukocyte Counting Rare Event Analysis Microorganism Analysis live dead others Fermentation control Particle Concentration Analysis True Volumetric Absolute Counting Particle Size and Fluorescence Distribution Analysis True Volumetric Absolute Counting Based on precise counting and m...

Page 42: ... all parameters or on specific trigger parameter selectable by software Data Resolution 65 536 channels 16 bit Service Please contact your local Sysmex representative 12 2 Optics Light Sources Laser Output Laser specifications may be subject to change Red Diode Laser 25 mW or 40 mW at 638 nm 640 nm Green solid state Laser 30 mW or 100 mW at 532 nm Blue Diode Laser 50 mW Blue solid state laser 30 2...

Page 43: ...ure for sheath fluid Sheath fluid pressure is adjustable from 0 300 mbar Computer controlled Default setting 200 mBar Sampling Volume Continuous up to 1000 µl 200 µl for precision absolute counting Free sampling volume with syringe counting method Flow Rates 1 Sample volume speed adjustable continuously between 0 and 20 µl s 2 Sheath fluid pressure continuously adjustable Fluidics Volume 2 x 2 lit...

Page 44: ...line Multi parameter gating Multicolour gating Peak and cluster analysis and statistics DNA cell cycle analysis DNA peak analysis Report module automated multi tube report generation as MS Word or MS Excel document Copy paste to desktop publishing software Ratio measurements Parameter arithmetic Acquisition Gating Lower level hardware thresholds for event triggering parameters adjusted by software...

Page 45: ...er with the acquisition data Report Generation Report module for Microsoft Word Excel directly called by the FloMax software Single or Multi Page Report templates include institute logo s addresses graphs and statistical results Templates can be individually adjusted in a desktop publishing manner Individual calculations can be defined in spread sheet formulas Copy Paste export to desktop publishi...

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