BD FACSCanto Reference Manual

Flow cytometer

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Page 1 BD FACSCanto Flow Cytometer Reference Manual For In Vitro Diagnostic Use bdbiosciences.com Part No. 642241 Rev. A June 2007 BD Biosciences Asia Pacific Brazil Canada 2350 Qume Drive Tel (65) 6-861-0633 Tel (55) 11-5185-9995 Toll Free (888) 259-0187 San Jose, CA 95131-1807...
Page 2 BD Biosciences. The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information.
Page 3 BD Biosciences, it is the buyer/user’s responsibility to install and maintain up-to-date virus protection software. BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation. BD Biosciences is not liable for any claims related to or resulting from buyer/user's failure to install and maintain virus protection.
Page 5: Table Of Contents
Computer Workstation ........BD FACS Loader (Optional) ....... . .
Page 6 Replacing the Fluidics Cart Fuses ......BD FACSCanto Flow Cytometer Reference Manual...
Page 7 Chapter 5: Troubleshooting Cytometer Troubleshooting ........Fluidics Cart Troubleshooting .
Page 8 Loader Specifications ......... . Index viii BD FACSCanto Flow Cytometer Reference Manual...
Page 9: About This Manual
BD Biosciences recommends that first-time users of this cytometer take advantage of operator training offered with the sale of every new cytometer. The BD FACSCanto Flow Cytometer Reference Manual assumes you have a working knowledge of basic Microsoft® Windows® operation. If you are not familiar with the Windows operating system, refer to the documentation provided with your computer.
Page 10: Conventions
Menu selection For example, “select File > Print” means select Print from the File menu. Ctrl-X Press the indicated keys simultaneously For example, Ctrl-P means hold down the Control key while pressing the letter p. BD FACSCanto Flow Cytometer Reference Manual...
Page 11: Technical Assistance
• error messages • details of system performance For cytometer support within the US, call (877) 232-8995. For support within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor. About This Manual...
Page 12 BD FACSCanto Flow Cytometer Reference Manual...
Page 13: Chapter 1: Introduction
The cytometer does not require special facilities—it plugs into a standard wall outlet, uses air-cooled lasers, and provides its own air pressure and vacuum for the fluidics and waste. You can prepare samples on the BD FACS™ Sample Prep Assistant II and import the sample prep worklist.
Page 14: Intended Use
Intended Use The BD FACSCanto system is intended for use as an In Vitro Diagnostic device for identification and enumeration of lymphocyte subsets in human cells in suspension for flow cytometry. System Components The BD FACSCanto system consists of a benchtop flow cytometer, a self- contained fluidics cart, and the BD FACSCanto workstation.
Page 15: Flow Cytometer
The BD FACSCanto flow cytometer consists of a fluidics subsystem, an optics subsystem, and an electronics subsystem. For a more in-depth discussion of Chapter 1: Introduction...
Page 16 (SIT) (Loader option only) aspirator arm aspirator arm bar BD FACSCanto Flow Cytometer Reference Manual...
Page 17 The following table briefly describes these components. flow cell where laser beam intercepts particles tubing brings sheath and cleaning fluids to and aspirates waste from flow cell hollow metal tube that brings sample to flow cell adapter lever lever used to change SIT from manual to automatic loading aspirator arm movable waste aspiration port aspirator arm...
Page 18 Optics The optics system in the BD FACSCanto cytometer is composed of both excitation optics and collection optics. Excitation optics bring light to the flow cell. Collection optics gather the light signals emitted or scattered by the particles.
Page 19 The BD FACSCanto cytometer uses low-powered air-cooled and solid state lasers that do not have special power and cooling requirements. Wavelength Laser Commonly Used Fluorochromes (nm) Coherent® Sapphire™ 488 (blue) FITC, PE, PerCP, Solid State PerCP-Cy™5.5, PE-Cy™7 JDS Uniphase™ HeNe...
Page 20 Figure 1-2 Optical pathway interlock flow cell prisms red He-Ne laser focusing lens blue solid-state laser When the flow cell access door opens, an interlock shutters the laser light and blocks its pathway for safety reasons. BD FACSCanto Flow Cytometer Reference Manual...
Page 21 Collection Optics From the flow cell, laser light is routed to the collection optics, which efficiently gather the signals emitted and scattered from each particle. The BD FACSCanto collection optics include two detector arrays, which consist of photomultiplier tubes (PMTs) arranged in one octagon and one trigon (Figure 1-3).
Page 22 In addition to the PMT detectors, a photodiode collects the stronger forward scatter signals. The obscuration bar prevents excess laser light from entering this diode (Figure 1-1 on page 18). For additional descriptions of detector arrays, mirrors, and filters, see Optics System on page 109. BD FACSCanto Flow Cytometer Reference Manual...
Page 23 At installation, the octagon and trigon arrays have the filter and mirror combinations shown in Table 1-1. Table 1-1 Octagon and trigon optical filters Detector Array BP Filter or LP Mirror Intended Dye (Laser) Position LP Mirror Octagon 780/60 PE-Cy7 (488-nm blue laser) PerCP-Cy5.5 or PerCP...
Page 24 On the BD FACSCanto, electronic system components consist of power controls and connectors along with processing boards in the card cage. This section describes only the user-accessible power panel.
Page 25: Fluidics Cart
Fluidics Cart The fluidics cart provides filtered sheath and cleaning fluids to the cytometer, and collects system waste products (Figure 1-5). The cart supplies the required air pressure and vacuum, which eliminates the need for an external source (although the cart can be hooked up to an in-house air source). Figure 1-5 Fluidics cart cubitainer 10-L waste...
Page 26 Containers and Ports The fluidics cart holds a 10-L waste container, a 20-L BD FACSFlow™ cubitainer, a 5-L BD FACS shutdown solution cubitainer, and a 5-L BD FACSClean solution cubitainer. Use the waste container provided with the system; do not substitute other containers.
Page 27 Leave the auxiliary air supply switch off unless the cart has been attached to an in-house air supply by BD Biosciences service personnel (see Table 1-2 for details). Leave the cart circuit breaker on at all times.
Page 28: Computer Workstation
BD FACS Loader (Optional) The BD FACS Loader automatically introduces prepared samples to the cytometer. The Loader is controlled by BD FACSCanto clinical software or BD FACSDiva software. It can be added to your system at any time. The Loader consists of: •...
Page 29 Figure 1-8 BD FACS Loader Chapter 1: Introduction...
Page 30: Barcode Reader (Optional)
The Opticon LG2 Imager is a hand-held barcode reader that plugs into the USB port on the BD FACSCanto computer workstation. A green light on the top of the barcode reader indicates that it is connected to the USB port and ready to use.
Page 31: System Requirements
• BD FACSDiva software v6.0 NOTICE BD FACSCanto clinical software v2.0 can be used to operate the BD FACSCanto system with BD FACSDiva™ software v5.0 installed. However, to use BD FACSDiva™ software v6.0, BD FACSCanto clinical software v2.1 must be installed.
Page 32 • full-strength bleach (waste tank) Fluids Required for External Cleaning • BD FACSClean solution • Deionized (DI) water Setup Beads BD FACS 7-color setup beads for use with BD FACSCanto clinical software BD FACSCanto Flow Cytometer Reference Manual...
Page 33: Chapter 2: Bd Facs Loader Option
BD FACS Loader Option This chapter contains the following information: • Loader Components on page 34 • Using the Loader on page 36 • Maintaining the Loader on page 43...
Page 34: Loader Components
(SIT) BD FACSCanto Flow Cytometer Reference Manual...
Page 35 The Loader is compatible with both the green-tinted carousels labeled “Sample- Prep Ready” and the traditional gray Loader carousels (Figure 2-2). Figure 2-2 Compatible carousels Sample-Prep Ready carousel traditional gray carousel Chapter 2: BD FACS Loader Option...
Page 36: Using The Loader
The Loader operates by software commands only. Commands can be issued with either BD FACSCanto clinical software or BD FACSDiva software. For instructions on running samples with the Loader, refer to the BD FACSCanto Instructions for Use. This section explains how to set up the cytometer for acquisition with the Loader.
Page 37 For accurate results, match the tubes to those listed on the printed worklist (BD FACSCanto clinical software) or the Carousel Assignment tab (BD FACSDiva software). Print a copy of tube assignments and use the printout as a guide when filling each carousel.
Page 38 To run the Loader, the cover must be in place on the drawer. Tubes will not be loaded if the cover is off. The currently running tube will be unloaded if the cover is removed during a run. Loader cover BD FACSCanto Flow Cytometer Reference Manual...
Page 39: Changing Operational Modes
Dispose of waste using proper precautions and in accordance with local regulations. Wear suitable protective clothing, eyewear, and gloves. Materials personal protective equipment Procedure Remove the Loader cover. Pull out the drawer. Remove any carousel in the Loader. Chapter 2: BD FACS Loader Option...
Page 40 Lift the adapter lever and rotate it into the highest position, as shown in the following figure. adapter lever, manual mode adapter lever, automatic mode (Optional) Rotate the aspirator arm bar vertically. automatic mode manual mode You are ready to load tubes manually. BD FACSCanto Flow Cytometer Reference Manual...
Page 41 Wear suitable protective clothing, eyewear, and gloves. Materials personal protective equipment Procedure Lift the adapter lever and rotate it into the automatic mode position, as shown in the following figure. adapter lever, manual mode adapter lever, automatic mode Chapter 2: BD FACS Loader Option...
Page 42 Move the tube guide arm all the way forward. The tube guide arm is needed only when using the Loader. automatic mode manual mode Pull out the drawer. You are ready to load tubes automatically. BD FACSCanto Flow Cytometer Reference Manual...
Page 43: Maintaining The Loader
Wear suitable protective clothing and gloves. To prevent damage, do not use isopropyl alcohol or ethanol on any Loader surfaces. Materials • BD FACSClean solution • DI water • clean, lint-free cloths or disposable wipes Chapter 2: BD FACS Loader Option...
Page 44 Switch off the cytometer power and unplug the AC power cord from the wall socket. Wipe all accessible surfaces with BD FACSClean solution. Ensure that the optical sensors are cleaned. Saline deposits can develop on the sensors, causing tubes in the carousel to be missed.
Page 45: Chapter 3: Barcode Reader Option
Barcode Reader Option This chapter contains the following information: • Installing and Using the Barcode Reader on page 46 • Cleaning the Barcode Reader on page 48 • Barcode Symbologies on page 49...
Page 46: Installing And Using The Barcode Reader
NOTICE The Opticon LG2 Imager2D barcode reader available for the BD FACSCanto is capable of reading both 1D and 2D barcode symbologies. See Barcode Symbologies on page 49. The BD FACSCanto system supports only two uses of the 2D barcode reader: •...
Page 47 Patient information is scanned from sample labels into the worklist ID field. Locate the barcode on the label of BD FACS 7-color setup beads kit, or on the patient sample. For accurate results, do not photocopy or enlarge the barcodes that are included with the reagent.
Page 48: Cleaning The Barcode Reader
• isopropyl alcohol • ethyl alcohol (denatured grade) Do not use BD FACSClean solution (or bleach) to clean or disinfect the barcode reader. BD FACSCanto Flow Cytometer Reference Manual...
Page 49: 1D Barcode Symbologies
By default, the barcode reader has checksums enabled. We recommend you do not disable checksums, or use barcode symbologies without checksums. 1D Barcode Symbologies BD Biosciences has evaluated the following 1D barcode symbologies for use with the BD FACSCanto flow cytometer, and has these recommendations: Barcode Symbology...
Page 50: 2D Barcode Symbologies
For information on installing and using the barcode readInstalling and Using the Barcode Reader on page 46er with the BD FACSCanto system, see Installing and Using the Barcode Reader on page 46. For detailed information including how to program the barcode reader for other barcode standards, refer to the information supplied by the manufacturer.
Page 51: Chapter 4: Maintenance
Maintenance The BD FACSCanto system requires basic preventive maintenance to preserve cytometer reliability. This section explains the procedures you should follow to keep your cytometer in good condition. • Scheduled Maintenance on page 52 • Unscheduled Maintenance on page 66...
Page 52: Scheduled Maintenance
Removes air from fluid filters, Weekly ensuring they will not dry out See Purging the Fluidics Filters on page 57. Change the waste tank See step 6 on page 56 (in Monthly Emptying the Waste Container procedure). BD FACSCanto Flow Cytometer Reference Manual...
Page 53 Decontaminates the internal Monthly and before (Long Clean) sheath path with service calls BD FACSClean solution, then rinses with BD FACS shutdown solution See Decontaminating the Fluidics System (Long Clean) on page 59. Replace the air filter Ensures proper cytometer...
Page 54: Emptying The Waste Container
Dispose of waste in accordance with local regulations. Use proper precaution and wear suitable protective clothing, eyewear, and gloves. waste port disposable waste cap trap waste tank Ensure the cytometer is not acquiring events. BD FACSCanto Flow Cytometer Reference Manual...
Page 55 Detach the waste container’s sensor and fluid line. • Pull the sensor straight out. • Press the metal clip on the fluid line. waste fluid line with metal clip sensor The waste tank can become pressurized when the cytometer is running. Always disconnect the tank from the fluidics cart before you empty it.
Page 56 Tighten each component only until it is hand-tight. Do not use sealants such as Teflon® tape or other adhesives. Screw the cap assembly onto the tank and hand-tighten until it is fully closed. Re-attach the sensor and fluid lines. BD FACSCanto Flow Cytometer Reference Manual...
Page 57: Purging The Fluidics Filters
Purging the Fluidics Filters Once a week, purge air from all fluid filters by opening the bleeder valve on the top of each filter, one at a time. This ensures that the filters will not dry out. NOTICE The cytometer must be on (system pressurized) during this procedure. Materials •...
Page 58 If the cubitainer contains fluid and the fluid lines are attached and primed, the filter might be airlocked. See Removing an Air Lock on page 72. Close the valve by turning it clockwise. Repeat steps 2 through 5 with the remaining filters. BD FACSCanto Flow Cytometer Reference Manual...
Page 59: Decontaminating The Fluidics System (Long Clean)
Decontaminating the Fluidics System (Long Clean) Use the Long Clean command to decontaminate the internal sheath path with BD FACSClean solution solution. After decontamination, the lines are rinsed with BD FACS shutdown solution. This procedure takes 75 minutes to complete.
Page 60: Replacing The Air Filter
If setup fails after a long clean, repeat Fluidics Startup until setup passes. Replacing the Air Filter The BD FACSCanto cytometer has an air filter located in the side door. Replace the filter every 6 months to ensure proper cytometer performance. Materials...
Page 61 • Twist the handle and pull. On the door’s interior, turn the pegs along the upper edge of the filter and remove the old filter. Chapter 4: Maintenance...
Page 62 Twist the handle while closing door. • Push door closed until the door latches. • Push the handle in to latch the handle. To avoid cytometer damage, do not close the door on any tubing or wires. BD FACSCanto Flow Cytometer Reference Manual...
Page 63: Replacing Fluidics Filters
Replacing Fluidics Filters Change the fluid filters when you see increased debris in FSC vs SSC plots, or every 6 months. filter Materials • replacement filter(s)—see Installation Kit on page 128 for ordering • paper towels • proper protective equipment •...
Page 64 Remove the filter by pressing the tabs on each quick-disconnect coupling. Figure 4-2 Removing the filter metal tab quick-disconnect coupling metal tab Position the new filter and connect the couplings. Write today’s date on the filter so you will know when to replace it. BD FACSCanto Flow Cytometer Reference Manual...
Page 65 Loosen the top bleeder valve by turning it counterclockwise. NOTICE Do not loosen the bottom bleeder valve. Ensure it is tightened. Do not completely unscrew the valve. If you do, it will come off. loosen this bleeder valve DO NOT loosen this bleeder valve Wait until fluid seeps out.
Page 66: Unscheduled Maintenance
See Cleaning the Flow Cell on needed page 77. Bubble Filter Purge Removes air from the bubble If fluidics run dry, or filter when CVs are poor See Purging the Bubble Filter on page 78. BD FACSCanto Flow Cytometer Reference Manual...
Page 67 Description and Location Frequency Decontaminate the Cleans out the fluidics lines with Before long-term storage fluidics system for BD FACSClean solution, then storage fills them with BD FACS shutdown solution See Decontaminating the Fluidics System for Storage on page 80.
Page 68: Changing A Cubitainer
Container Port Label Port Color waste Waste (A) orange-red BD FACSFlow solution BD FACSFlow blue BD FACS shutdown solution BD FACS Shutdown Solution green BD FACSClean solution BD FACS Cleaning Solution green BD FACSCanto Flow Cytometer Reference Manual...
Page 69 Procedure Ensure the cytometer is not acquiring events. Detach the sensor and fluid line from the cart. • Pull the sensor straight out. sensor • Press the metal clip on the fluid line. metal clip You could damage the sensor line if you leave it connected while changing a cubitainer.
Page 70 To ensure that the appropriate solutions are dispensed, match the label on the container to the port on the fluidics cart. Prime the fluidics. Important: Continue with the procedure in Priming Fluidics Lines on page 71. BD FACSCanto Flow Cytometer Reference Manual...
Page 71 Select Cytometer > Cleaning Modes > Prime After Tank Refill. Select the checkboxes for the cubitainers you changed; click OK. BD FACSCanto clinical software BD FACSDiva software Click OK when the completion message displays.
Page 72: Removing An Air Lock
• bypass tubing Procedure Place a few paper towels beneath the air-locked filter to collect drips. Remove the filter by pressing the metal tabs. Figure 4-3 Removing a filter metal tab metal tab BD FACSCanto Flow Cytometer Reference Manual...
Page 73 Select the checkbox that corresponds to the filter you have bypassed. Click OK. BD FACSDiva software BD FACSCanto clinical software When the prime finishes, remove the bypass tubing. Reattach the filter to the fluidics cart. Repeat the Prime After Tank Refill command.
Page 74 Open the bleeder valve and wait for fluid to seep out. Close the valve. If no fluid seeps out, repeat steps 9 and 10. Figure 4-4 Bleeder valve loosen this bleeder valve DO NOT loosen this bleeder valve BD FACSCanto Flow Cytometer Reference Manual...
Page 75: Cleaning External Surfaces
80. NOTICE Do not use BD FACSClean solution or bleach to clean or disinfect the barcode reader. See Cleaning the Barcode Reader on page 48. All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease.
Page 76: Removing Bubbles From The Flow Cell
To avoid potential shock, always switch off the power and unplug the AC power cord before you begin cleaning. Wipe all accessible surfaces with BD FACSClean solution. Wet a fresh cloth with DI water and wipe all bleach-exposed surfaces to prevent corrosion.
Page 77: Cleaning The Flow Cell
Cleaning the Flow Cell Use the Clean Flow Cell command to run a tube of BD FACSClean solution through the SIT and flow cell. After the procedure is complete, the cleaning fluid remains in the SIT and flow cell until you run fluidics startup or shutdown.
Page 78: Purging The Bubble Filter
A progress message displays during the cleaning. BD FACSCanto clinical software BD FACSDiva software After the completion message displays, wait 5 minutes. Waiting allows the BD FACSClean solution to dissolve deposits in the flow cell cuvette. Click OK. Remove the tube from the SIT.
Page 79 A dialog indicates Bubble Filter Purge progress. Wait until the purge finishes. BD FACSDiva software BD FACSCanto clinical software Click OK when the completion message is displayed. BD FACSCanto clinical software BD FACSDiva software Repeat steps 1 through 3 until bubble-free liquid enters the flow cell from the bubble filter.
Page 80: Decontaminating The Fluidics System For Storage
Decontaminating the Fluidics System for Storage If your cytometer is to be taken out of operation, perform the following procedure to clean out the fluidics lines and fill them with BD FACS shutdown solution. Perform steps 1 through 4 in Decontaminating the Fluidics System (Long Clean) on page 59.
Page 81 Materials • proper protective equipment • replacement Bal seal • DI water Procedure Turn off the cytometer. If you have a Loader, follow these steps: • Remove the Loader cover. • Pull out the drawer. • Remove any carousel. • Change the cytometer from automatic to manual mode (see Changing to Manual Loading on page 39).
Page 82 Move the aspirator arm to the left and hold it. aspirator arm bar aspirator arm Remove the retainer from the SIT by turning it in the direction shown. BD FACSCanto Flow Cytometer Reference Manual...
Page 83 Remove the Bal seal by pulling it off with your fingers. Bal seal Install the new Bal seal spring-side up. Chapter 4: Maintenance...
Page 84: Resetting The Cytometer Circuit Breaker
Test by manually loading a tube onto the SIT and running fluid. Resetting the Cytometer Circuit Breaker Toggle the cytometer circuit breaker switch to on. main power button cytometer circuit breaker, on If it is off, turn on the cytometer main power. BD FACSCanto Flow Cytometer Reference Manual...
Page 85: Reconnecting The Ethernet And Rs232 Cables
Reconnecting the Ethernet and RS232 Cables The cytometer connects to and communicates with the workstation through Ethernet and RS232 network cables. If these cables should become disconnected, use the following diagram to reconnect them. Figure 4-6 Ethernet and RS232 network cable RS232 cable Ethernet cable Both cables connect to ports on the PC workstation.
Page 86 Power In c. Air In m. Air Out d. Waste (A) o. Waste e. Communications j. Communication f. Sheath (B) i. Fluid Out g. Waste (A) n. Waste — h. On/Off — l. Air In BD FACSCanto Flow Cytometer Reference Manual...
Page 87 70±5 psi d. Waste (A) Waste out (aspirated) e. Communications Data port f. Sheath (B) BD FACSFlow solution port g. Waste (A) Waste out (non-aspirated) h. On/Off Auxiliary air supply switch. Keep in off position unless connected to house air.
Page 88: Replacing The Fluidics Level Sensors
Replacing the Fluidics Level Sensors Replace the fluidics level sensors when instructed to do so by a BD Biosciences service representative. Before you replace a sensor, try rinsing it with DI water. If you are changing the sensor on the waste tank, use proper precaution and wear suitable protective clothing, eyewear, and gloves.
Page 89 Procedure Ensure that the cytometer is not acquiring events. Detach the sensor and fluid line from the cart. Unscrew the cap on the cubitainer. Remove the level sensor assembly and discard into a suitable receptacle. Chapter 4: Maintenance...
Page 90 If you replaced any level sensors (other than the waste sensor), prime the affected fluidics lines. Important: Continue with the procedure in Priming Fluidics Lines on page 71. BD FACSCanto Flow Cytometer Reference Manual...
Page 91: Replacing The Fluidics Cart Fuses
Replacing the Fluidics Cart Fuses Power surges and other electrical events could cause a fuse to blow. Use the following procedure to replace the fuses. Materials • two replacement fuses (see Installation Kit on page 128 for ordering) • small screwdriver Removing the Old Fuses To protect against shock, always turn off the cytometer and unplug the power cord before performing this procedure.
Page 92 Remove the fuse drawer, and remove and dispose of the old fuses. Note the positions of the old fuses before you remove them. Duplicate these positions with the new fuses. Replace both fuses. Make sure the fuse sits as shown: right wrong BD FACSCanto Flow Cytometer Reference Manual...
Page 93 For protection against risk of fire, replace fuses only with those provided by BD Biosciences. Make sure the text for your area’s voltage is right-side up. Slide the drawer back into the cytometer until it snaps into place. Close the fuse access door.
Page 94 BD FACSCanto Flow Cytometer Reference Manual...
Page 95: Chapter 5: Troubleshooting
Troubleshooting The tips in this section can help you troubleshoot issues that might arise when using this cytometer. For software troubleshooting, refer to the BD FACSCanto Clinical Software Reference Manual, the BD FACSDiva Software Reference Manual, or the BD FACSCanto Instructions for Use.
Page 96: Cytometer Troubleshooting
Air in BD FACSFlow filter Purge air from the filter. See (fluidics cart) Purging the Fluidics Filters on page 57. Air lock in BD FACSFlow See Removing an Air Lock on filter (on fluidics cart) page 72. BD FACSCanto Flow Cytometer Reference Manual...
Page 97 Bal seal improperly installed Reinstall or replace the Bal seal. or worn See Replacing the Bal Seal on page 80. Air lock in BD FACSFlow See Removing an Air Lock on filter page 72. Liquid leakage around Interior valve failure Turn off the cytometer power.
Page 98 Cytometer circuit breaker Switch on the cytometer circuit tripped breaker, followed by the cytometer power (if off). Blown fuse in cytometer Turn off the cytometer main controller power and call BD Biosciences. BD FACSCanto Flow Cytometer Reference Manual...
Page 99 Use Falcon brand 12 x 75-mm sample tube does not fit polystyrene test tubes, snugly on SIT BD Trucount tubes, or BD FACS 7-color setup bead tubes. See System Requirements on page 31. Bal seal improperly installed Reinstall or replace the Bal seal.
Page 100 Call BD Biosciences. Waste line to fluidics cart Turn off the cytometer power. disconnected Clean up the liquid, using proper precautions. Check that waste lines are securely plugged in. Turn on the cytometer power. BD FACSCanto Flow Cytometer Reference Manual...
Page 101: Fluidics Cart Troubleshooting
Clean up the liquid. Check and close all bleeder valves. See Purging the Fluidics Filters on page 57. Broken fluid line Contact BD Biosciences. Fluidics cart will not Cart circuit breaker off Switch on the cart circuit breaker. power on, cytometer on Auxiliary air supply on, cart Toggle the auxiliary air supply off.
Page 102: Loader Troubleshooting
BD FACSDiva software. Refer to sample the BD FACSCanto Instructions for Use. Bubble in flow cell diverts Degas flow cell. See Removing stream Bubbles from the Flow Cell on page 76. BD FACSCanto Flow Cytometer Reference Manual...
Page 103 See step 4 on page 38. If the problem persists, contact BD Biosciences for assistance. Aspirator arm bar vertical Rotate the bar horizontally. See Changing to Automatic Loading on page 41.
Page 104 BD FACSCanto Flow Cytometer Reference Manual...
Page 105: Appendix A: Technical Overview
Appendix A Technical Overview This appendix provides more information about these topics. • Flow Cytometry on page 106 • Fluidics System on page 106 • Optics System on page 109 • Electronics System on page 120...
Page 106: Flow Cytometry
Sheath Cubitainer to Flow Cell On the BD FACSCanto flow cytometer, a separate fluidics cart houses the sheath cubitainer, cleaning fluid cubitainers, and the waste tank. Positive-pressure pumps in the cart send sheath fluid past a 0.22-µm filter to a pressurized interior reservoir within the cytometer, called the plenum.
Page 107 Figure A-1 Sheath and sample flow pathways for the BD FACSCanto cytometer Sheath Flow Sample Flow sheath tank test tube sheath filter interior reservoir (plenum) sample injection tube (SIT) bubble filter flow cell interrogation point waste aspirator waste tank You can view the current sheath fluid pressure in BD FACSCanto clinical software by displaying the Status window.
Page 108 A higher flow rate is generally used for measurements such as immunophenotyping. The data is less resolved but is acquired more quickly. • A lower flow rate is generally used in applications where greater resolution is critical. BD FACSCanto Flow Cytometer Reference Manual...
Page 109: Optics System
When using BD FACSCanto clinical software, the cytometer automatically regulates the sample pressure according to the currently selected panel. Optics System As stained cells or other particles pass through the focused laser beam, they scatter the laser light and fluoresce.
Page 110 Figure A-3 Light pathway through the BD FACSCanto flow cytometer stained particle prisms forward scatter collecting focusing fiber optic cables diode detector lens lens obscuration steering plate detector mirror flow cell red laser fiber optic cable filter blue laser BD FACSCanto Flow Cytometer Reference Manual...
Page 111: Light Scatter
Light Scatter When a cell or particle passes through a focused laser beam, laser light is scattered in all directions (Figure A-4). Light that scatters roughly in the same direction as the laser beam is called forward scatter (FSC); light that scatters roughly perpendicular to the laser beam is called side scatter (SSC).
Page 112: Fluorescence
The emission spectra for some commonly used fluorochromes are shown in Figure A-5. Figure A-5 Emission spectra of commonly used fluorochromes 100% FITC PerCP-Cy5.5 PE-Cy7 APC-Cy7 PE-Cy7 APC-Cy7 wavelength (nm) BD FACSCanto Flow Cytometer Reference Manual...
Page 113: Optical Filters
Figure A-6 Effect of an optical filter on incident photons photons absorbed photons photons reflected transmitted Two kinds of filters are used on the BD FACSCanto flow cytometer (default configuration): • longpass (LP) • bandpass (BP) Longpass Filter In general, LP filters pass wavelengths longer than the filter rating. For example,...
Page 114 For example, a 500/50 BP filter transmits light that is centered at 500 nm and has a total bandwidth of 50 nm. Therefore, this filter transmits light efficiently between 475 and 525 nm. BD FACSCanto Flow Cytometer Reference Manual...
Page 115: Detectors
Detectors convert light signals into electrical signals that can be processed by the electronics system and a computer and then displayed on a plot. There are two types of signal detectors in the BD FACSCanto flow cytometer: the photodiode and the photomultiplier tube (PMT). The photodiode is used to detect the stronger FSC signal (generated by light from the blue laser).
Page 116 Behind the LP mirror, a 780/60 BP filter admits light from 750 to 810 nm and substantially blocks other wavelengths. The light that finally reaches PMT A will be from dyes such as PE-Cy7 that emit in this range. BD FACSCanto Flow Cytometer Reference Manual...
Page 117 Figure A-10 Excitation and emission spectra of PE-Cy7 blue laser bandpass excitation light filter emission Likewise, the 655 LP mirror for PMT B will reflect light with a wavelength of less than 655 nm to the next PMT. Light with a longer wavelength will pass through to another LP filter (670 nm) that further blocks shorter wavelength light.
Page 118: Spillover
Spillover The arrangement of filters and mirrors in the BD FACSCanto enables each PMT to receive signals predominantly from a specific fluorochrome. However, fluorochromes emit light over a range of wavelengths. As a result, a signal from one fluorochrome can appear in a detector used for another fluorochrome.
Page 119 The software automatically determines these compensations during setup, which you can also manually adjust: • In BD FACSCanto clinical software, adjust spectral overlap on the Spectral Overlap tab. • In BD FACSDiva software, adjust spectral overlap in the Compensation tab in the Cytometer window.
Page 120: Electronics System
18 binary digits that are either 1 or 0. 100100101011101010 is an example of an 18-bit number. Converted into base 10, the scale we normally use, this number is 150,250. There are 262,144 18-bit values. BD FACSCanto Flow Cytometer Reference Manual...
Page 121: Pulses
Pulses Inside the PMTs, the laser light is converted into an electrical signal. This electrical signal is called a pulse. Figure A-15 Anatomy of a pulse laser beam particle time time time A pulse begins when a particle enters the laser beam. At this point, both the beam intensity and signal intensity are low.
Page 122: Pulse Measurements
(ADC) that changes the analog (continuous) pulse into digital (discrete) data. The ADC does this by sampling the pulse up to 10 million times per second, slicing it into 16,384 levels, and assigning a measurement to each time sample (Figure A-17 on page 123). BD FACSCanto Flow Cytometer Reference Manual...
Page 123 Figure A-17 A digitized pulse 10,000,000 measurements per second measurements digitized into 16,384 levels (14 bits) The ADC sends these numbers to a programmable digital delay, so that the numbers can be adjusted in time to account for the spatial displacement of the two excitation laser beams (blue and red).
Page 124 Because data has been converted into 18-bits, an 18-bit display is used to keep all data on scale. That means a pulse (an event), will fall into one of 262,144 digital bins, or channels, when it is eventually assigned to a dot plot or histogram. BD FACSCanto Flow Cytometer Reference Manual...
Page 125 The electronics boards include an embedded computer that sends pulse measurements and other data from the cytometer to your workstation. For more about digital theory, refer to Appendix B in the BD FACSDiva Software Reference Manual. For an in-depth discussion, visit our website at bdbiosciences.com/immunocytometry_systems/ and download the BD FACSDiva...
Page 126 BD FACSCanto Flow Cytometer Reference Manual...
Page 127: Appendix B: Supplies And Replacement Parts
Appendix B Supplies and Replacement Parts To order supplies and options, contact your local BD Biosciences representative. For updated information and part numbers, refer to our website bdbiosciences.com. • Cytometer Supplies on page 128 • Consumables on page 130...
Page 128: Cytometer Supplies
10-L waste tank 340261 Vented cap for waste tank (pack of 12) 338922 Waste cap baffle 338505 Spare fluidics cart fuses (2): 6.3-A 250V Slo-blo 90069-24 Type T Loader cover (Loader option only) 338505 BD FACSCanto Flow Cytometer Reference Manual...
Page 129: Other Replacement Parts
Other Replacement Parts Item Part No. Sheath sensor probe (5-level) 334914 Waste sensor probe (6-level) 334915 Auxiliary sensor probe (1-level) 334911 Air filter (side door) 336303 Bypass tubing 336768 Bal seal retainer 336999 Carousel kit, carousels 1–4 332727 Carousel kit, carousels 5–8 332728 Carousel kit, carousels 9–12 332729...
Page 130: Consumables
334224 Monoclonal antibodies BD Biosciences BD FACS™ lysing solution BD Biosciences 349202 a. Refer to the BD Biosciences Immunocytometry Products Catalog or the BD Biosciences website (bdbiosciences.com). b. US Patent Nos. 4,654,312; 4,902,613; 5,098,849 BD FACSCanto Flow Cytometer Reference Manual...
Page 131: Labware
Labware Item Supplier Catalog No. 5-mL polystyrene test tubes, BD Biosciences 12 x 75-mm (BD Falcon tubes) (877) 232-8995 • uncapped, 125 per bag • 352052 • capped, 125 per bag • 352054 • capped, 25 per bag • 352058 •...
Page 132 BD FACSCanto Flow Cytometer Reference Manual...
Page 133: Appendix C: Technical Specifications
Appendix C Technical Specifications • Cytometer Specifications on page 134 • Fluidics Cart Specifications on page 138 • Loader Specifications on page 139 For barcode reader specifications, refer to the information supplied by the manufacturer.
Page 134: Cytometer Specifications
149.7 kg (330 lb)—cytometer only, excluding Loader and computer Maximum 167.8 kg (370 lb)—including Loader Power requirements 100/115/230 VAC (50–60 Hz) Current: 5A at 115 VAC 2.5A at 230 VAC Power consumption 500 W BD FACSCanto Flow Cytometer Reference Manual...
Page 135: Environment
Sensitivity enables the resolution of platelets from noise Forward scatter sensitivity 1 micron Side scatter sensitivity 0.5 micron Optics Laser Specifications The following Class 3B lasers are mounted on the BD FACSCanto cytometer. Wavelength Power Manufacturer Model (nm) (mW) Coherent...
Page 136 • 515–545 nm (FITC) Two wavelength ranges detected from 633-nm laser: • 750–810 nm (APC-Cy7) • 650–670 nm (APC) Forward scatter detection Photodiode with 488/10 bandpass filter Side scatter detection PMT with 488/10 bandpass filter BD FACSCanto Flow Cytometer Reference Manual...
Page 137: Fluidics
Sheath consumption 1.1 L/hr, normal operation <1.0 mL/hr, standby Sample flow rates Assay dependent, controlled automatically by BD FACSCanto clinical software. Nominal rates: Low = 10 µL/min Medium = 60 µL/min High = 120 µL/min Sample acquisition rate 10,000 events/sec with <10% abort rate Recommended maximum particle 50 µm...
Page 138: Fluidics Cart Specifications
45.4 kg (≤100 lb)—fluidics cart only, excluding tanks 81.7 kg (≤180 lb)—with tanks full Facilities No air supply or vacuum required Capacity BD FACSFlow cubitainer 20 L BD FACSClean solution cubitainer BD FACS shutdown solution cubitainer Waste tank 10 L BD FACSCanto Flow Cytometer Reference Manual...
Page 139: Loader Specifications
Accommodates up to 40 uncapped 12 x 75-mm tubes • BD Falcon polystyrene test tubes • BD Trucount tubes • BD FACS 7-color setup bead tubes • Thickness of accumulated labels ≤5 mils (0.125 mm) no more than 3 labels thick ≤1.07 mL...
Page 140 BD FACSCanto Flow Cytometer Reference Manual...
Page 141: Index
48 SIT 77 installing 46 compensation theory 118–120 barcode symbologies 49 components bleeding filters 57 FACSCanto system 14 breaker, main circuit 24 fluidics cart 25 Bubble Filter Purge 66, 78 Loader 34 button, power 24 connecting power cords 24...
Page 142 75 dimensions 138 fuses, replacing 91 maintenance 54, 68 reconnecting tubing 85 fiber optics 20 troubleshooting 98, 101 fluids leaks cytometer 97 fluidics cart 101 SIT 100 not recommended 75 required 31 fluorescence 112 BD FACSCanto Flow Cytometer Reference Manual...
Page 143 fluorochromes lifter, tube 34 emission spectra 112 Loader for 488-nm and 633-nm lasers 19 about 28 spectral overlap and 118 adapting cytometer 36, 41 focusing lens 20 cleaning 43 forward scatter (FSC) components 34 about 111 long clean 59 diode 18 longpass (LP) filters 113 fuses, replacing 91 maintenance...
Page 144 111 scheduled maintenance 52 waste container 26 seal, Bal 80 cap 55 sensors, fluidics 88 emptying 54 side door, cytometer 15 side scatter (SSC) 111 specifications 139 cytometer 134 fluidics cart 138 spindle 35 BD FACSCanto Flow Cytometer Reference Manual...

BD FACSCanto Reference Manual

About this Manual

Chapter 1: Introduction

Chapter 2: BD FACS Loader Option

Chapter 3: Barcode Reader Option

Chapter 4: Maintenance

Chapter 5: Troubleshooting

Appendix A: Technical Overview

Appendix B: Supplies and Replacement Parts

Appendix C: Technical Specifications

Index

Quick Links

Troubleshooting

Need help?

Questions and answers

Related Manuals for BD FACSCanto

Summary of Contents for BD FACSCanto

Table of Contents