BD FACSCanto Flow Cytometer Reference Manual

BD FACSCanto Flow Cytometer Reference Manual
For In Vitro Diagnostic Use

bdbiosciences.com Part No. 642241 Rev. A June 2007
BD Biosciences 2350 Qume Drive San Jose, CA 95131-1807 USA Tel (877) 232-8995 Fax (800) 325-9637 [email protected]
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2007, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from 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. BD Biosciences welcomes customer input on corrections and suggestions for improvement. BD FACSDiva software Becton, Dickinson and Company. This software is the property of Becton, Dickinson and Company. Each sale of a stored unit of this software grants the purchaser a nontransferable, nonexclusive, personal license. This software may not be duplicated, reproduced, or copied in any form or by any means whatsoever, except as otherwise permitted by law. BD FACSCanto clinical software Becton, Dickinson and Company. This software is the property of Becton, Dickinson and Company. Each sale of a stored unit of this software grants the purchaser a nontransferable, nonexclusive, personal license. This software may not be duplicated, reproduced, or copied in any form or by any means whatsoever, except as otherwise permitted by law. BD, BD logo, and all other trademarks are property of Becton, Dickinson and Company. Cy is a trademark of Amersham Biosciences Corp. Cy dyes are subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp. only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp., 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA. JDS Uniphase is a trademark of JDS Uniphase, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation. Sapphire is a trademark and Coherent is a registered trademark of COHERENT, INC. Teflon is a registered trademark of E.I. du Pont de Nemours and Company. All other company and product names might be trademarks of the respective companies with which they are associated.
Patents
PerCP: US 4,876,190 APC-Cy7: US 5,714,386
FCC Information
WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the users authority to operate the equipment. NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, can cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense. Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits. This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations. Cet appareil numrique de la classe A respecte toutes les exigences du Rglement sur le matriel brouilleur du Canada.
Notice
BD Biosciences delivers software and workstations that are intended for running the cytometers supplied by BD Biosciences. It is the responsibility of the buyer/user to ensure that all added electronic files including software and transport media are virus free. If the workstation is used for Internet access or purposes other than those specified by BD Biosciences, it is the buyer/users 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.
History
Revision 336928 Rev. A 337969 Rev. A 338619 Rev. A 343370 Rev. A 640801 Rev. A 642241 Rev. A Date 1/04 4/04 9/04 9/05 5/06 6/07 Change Made Initial release Updated for CE IVD release Updated for BD FACSDiva software v4.1 Updated for BD FACSCanto clinical software v2.0 and the Opticon LG2 Imager barcode reader Updated for BD FACSDiva software v5.0 and BD FACSCanto clinical software v2.1 Updated for BD FACSDiva software v6.0
Contents
About This Manual Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 1: Introduction Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluidics Cart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Computer Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BD FACS Loader (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Barcode Reader (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 2: BD FACS Loader Option Loader Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changing Operational Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maintaining the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleaning External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix x xi 13 14 14 15 25 28 28 30 31 33 34 36 39 43 43
Chapter 3: Barcode Reader Option Installing and Using the Barcode Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleaning the Barcode Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1D Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2D Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 4: Maintenance Scheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emptying the Waste Container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purging the Fluidics Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Decontaminating the Fluidics System (Long Clean) . . . . . . . . . . . . . . . . Replacing the Air Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replacing Fluidics Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unscheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changing a Cubitainer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Removing an Air Lock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleaning External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Removing Bubbles from the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . Cleaning the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purging the Bubble Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Decontaminating the Fluidics System for Storage . . . . . . . . . . . . . . . . . . Replacing the Bal Seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resetting the Cytometer Circuit Breaker . . . . . . . . . . . . . . . . . . . . . . . . . Reconnecting the Ethernet and RS232 Cables . . . . . . . . . . . . . . . . . . . . . Reconnecting the Fluidics Cart Tubing . . . . . . . . . . . . . . . . . . . . . . . . . . Replacing the Fluidics Level Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replacing the Fluidics Cart Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45 46 48 49 49 50 51 52 54 57 59 60 63 66 68 72 75 76 77 78 80 80 84 85 85 88 91
vi
Chapter 5: Troubleshooting Cytometer Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluidics Cart Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loader Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A: Technical Overview Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluidics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spillover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electronics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulse Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix B: Supplies and Replacement Parts Cytometer Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Installation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Replacement Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bar Code Reader Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytometer Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
95 96 101 102 105 106 106 109 111 112 113 115 115 118 120 121 122 127 128 128 129 129 130 130 130 131
Contents
vii
Appendix C: Technical Specifications Cytometer Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signal Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluidics Cart Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loader Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index
133 134 135 135 135 137 137 138 138 139 141
viii
About This Manual

This manual contains reference information about the BD FACSCanto flow cytometer and BD FACS Loader. Operating instructions are contained in the BD FACSCanto Instructions for Use. Most cytometer functions are controlled by BD FACSCanto clinical software and BD FACSDiva software. BD FACSCanto clinical software contains modules for dedicated clinical applications with automatic gating algorithms, while BD FACSDiva software is nonapplication specific. You can use either software to perform cytometer quality control. Each software package has its own reference manual, provided on the documentation CD. 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.
ix
Conventions
The following tables list conventions used in this manual. Table 1 lists symbols that are used in this manual or on safety labels to alert you to a potential hazard. Text and keyboard conventions are shown in Table 2.
Table 1 Hazard symbolsa
Symbol Meaning CAUTION: hazard or unsafe practice that could result in material damage, data loss, minor or severe injury, or death Electrical danger Laser radiation Biological risk
a. Although these symbols appear in color on the cytometer, they are in black and white throughout this reference manual; their meaning remains unchanged.
Table 2 Text and keyboard conventions

Convention Use Features or hints that can save time or prevent difficulties Provides information that supplements the topic material Book titles and new or unfamiliar terms 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.
Tip NOTICE
Italics >
Technical Assistance
For technical questions or assistance in solving a problem: Read the section of the manual specific to the operation you are performing. See Chapter 5, Troubleshooting.
If additional assistance is required, contact your local BD Biosciences technical support representative or supplier. When contacting BD Biosciences, have the following information available: product name, part number, and serial number software version number 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
xi
xii
1
Introduction
The BD FACSCanto system uses a fixed-optics design and advanced digital electronics to support multicolor analysis of up to six fluorescent markers and two scatter parameters per assay. The cytometer does not require special facilitiesit 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. For further automation, use BD FACSCanto clinical software and the BD FACS Loader for sample acquisition. Alternatively, use BD FACSDiva software for more flexibility in acquisition and analysis. See the following for more information: Intended Use on page 14 System Components on page 14 System Requirements on page 31
13
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 selfcontained fluidics cart, and the BD FACSCanto workstation. System options include an automated sample loader and a barcode reader.
For a description of system components, see: Flow Cytometer on page 15 Fluidics Cart on page 25
14
Computer Workstation on page 28 BD FACS Loader (Optional) on page 28 Barcode Reader (Optional) on page 30
Flow Cytometer
Cytometer power is controlled by the power button. All other cytometer and fluidics cart functions are controlled by BD FACSCanto clinical software and BD FACSDiva software (provided with the cytometer).
flow cell access door side door data ports optics access door
sample injection tube
power button fluidics cart connections
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
15
fluidics, optics, electronics, and flow cytometry, see the Technical Overview on page 105.
Fluidics
The fluidics system consists of the sample injection tube (SIT), the aspirator arm, the flow cell, a pressurized interior reservoir, and a network of tubing that provides sheath and cleaning fluids to and removes waste from the flow cell.
flow cell
tubing
adapter lever sample injection tube (SIT)
tube guide (Loader option only) aspirator arm bar
aspirator arm
16
The following table briefly describes these components.

flow cell tubing SIT adapter lever aspirator arm aspirator arm bar tube guide where laser beam intercepts particles brings sheath and cleaning fluids to and aspirates waste from flow cell hollow metal tube that brings sample to flow cell lever used to change SIT from manual to automatic loading movable waste aspiration port metal bar used to push aspirator arm away from SIT during manual loading helps guide tube onto the SIT (Loader operation only)
When you install tubes onto the SIT, a pump within the fluidics cart pressurizes the interior reservoir (plenum), which then provides sheath fluid to the flow cell. At the same time, the sample tube is pressurized and sample is pushed up the SIT and into the flow cell. When you remove tubes from the SIT, the cytometer cleans the SIT by flushing sheath solution down the inside and outside of the tube. The flushed sheath solution is aspirated by the aspirator arm. To activate SIT cleaning, push the aspirator arm all the way to the left when you manually unload a tube. When you are using the Loader, SIT cleaning occurs automatically. Do not leave a tube of distilled water on the SIT between sample tubes, or during or after daily shutdown.
17
Flow Cell
Once the sample moves into the flow cell, particles move in single file past the laser beams. The scattered and emitted light from these particles provides information about their size, shape, granularity, and fluorescence properties. The flow cell is beneath the flow cell access door. For more information, see Fluidics System on page 106.
Figure 1-1 Flow cell
where lasers intercept sample stream
obscuration bar FSC diode
flow cell
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.
Excitation Optics
The excitation optics consist of lasers, fiber optic cables, beam-shaping prisms, and an achromatic focusing lens, as shown in Figure 1-2 on page 20.
18
The BD FACSCanto cytometer uses low-powered air-cooled and solid state lasers that do not have special power and cooling requirements.
Wavelength (nm) 488 (blue) 633 (red)
Laser Coherent Sapphire Solid State JDS Uniphase HeNe Air Cooled
Commonly Used Fluorochromes FITC, PE, PerCP, PerCP-Cy5.5, PE-Cy7 APC, APC-Cy7
19
Fiber optic cables direct the laser light onto beam-shaping prisms, which in turn transmit the laser light to a focusing lens. The lens directs the laser light onto the sample stream within the flow cell (Figure 1-2). See also Figure A-3 on page 110.
Figure 1-2 Optical pathway
1 3 2
1 2 3
interlock prisms focusing lens
4 5 6
flow cell red He-Ne laser blue solid-state laser
When the flow cell access door opens, an interlock shutters the laser light and blocks its pathway for safety reasons.
20
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). The octagon contains five PMTs and detects light from the 488-nm (blue) laser. One PMT in the octagon collects side scatter (SSC) signals. The trigon contains two PMTs and detects light from the 633-nm (red) laser.
Figure 1-3 Octagon and trigon detector arrays
red-laser signal
H
B
A
78
73 5L
660/20
0/6 0
trigon
530/30
octagon
502LP
585/4 2
556LP
67
longpass mirror bandpass filter

A
780
P 0L
655
LP
PMT
blue-laser signal
/60
735
LP
488/1 0
21
When light arrives at an array, a longpass mirror (filter) transmits the highest wavelengths to the first PMT in the series and reflects lower wavelengths to the next PMT. Similarly, the next PMTs longpass mirror transmits the next highest wavelengths and reflect lower wavelengths, and so on around the array. A bandpass filter (or additional longpass mirror) in front of each PMT further screens unwanted light.
A
B
Fiber
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.
22
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 (Laser) Octagon (488-nm blue laser) PMT Position A B C D E F G H Trigon (633-nm red laser) A B LP Mirror 735 655 556 502 blank optical holder blank optical holder blank optical holder 735 blank BP Filter or LP Mirror 780/60 670 585/42 530/30 488/10 blank optical holder blank optical holder blank optical holder 780/60 660/20 Intended Dye PE-Cy7 PerCP-Cy5.5 or PerCP PE FITC SSC APC-Cy7 APC
Blank optical holders do not contain optical filters. They are used in the octagon and trigon to prevent unwanted light from interfering with fluorescence signal.
23
Electronics
The electronics system converts optical signals to electronic signals and digitizes them for computer analysis. The photodiode and PMTs generate signals proportional to the amount of light they detect. The cytometers onboard electronics amplifies and then converts the signals from continuous voltage values (analog) into discrete values (digital). Upon amplification and digital conversion, fluorescent light signals from consistently prepared and stained particles characteristically fall into certain channels, thus allowing analysis. 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. For more information, see Electronics System on page 120.
Power Panel
Power to the cytometer, lasers, and fluidics cart is supplied by a power cord from the cytometer plugged directly into a standard electrical outlet. BD recommends using an uninterrupted power supply (UPS) unit to maintain cytometer power during a power outage. The system power button turns on the cytometer and fluidics cart, and powers the lasers (Figure 1-4).
Figure 1-4 Flow cytometer power panel
system power button
system AC power cord plugs in here
system circuit breaker
The system circuit breaker is located next to the AC power cord. The breaker will need to be reset if there is a power surge in the laboratory.
24
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 container pressure gauge door access knob
filters
25
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.
Figure 1-6 Fluidics cart containers and ports
BD FACS shutdown solution port and cubitainer BD FACSClean port and cubitainer
BD FACSFlow port waste port
waste tank
BD FACSFlow cubitainer
Each solution has its own non-interchangeable fluid port and level-sensor connection. Fluid level alarms occur within BD FACSCanto clinical software and BD FACSDiva software.
26
Controls
The fluidics cart connects to the flow cytometer unit by way of cables and tubing. When you turn on the power to the cytometer, the fluidics cart powers on also. Under ordinary circumstances, you do not need to adjust any of the switches on the carts power panel. 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.
Figure 1-7 Fluidics cart panel
auxiliary air supply switch, off
cart circuit breaker, on
Table 1-2 Auxiliary air supply switch positions

Position Off On Air Source Cart air House air What This Means The cart is providing its own air pressure and vacuum. The cart is hooked up to the buildings air pressure and vacuum.
27
Powering Off
To turn off the fluidics cart (and the cytometer, as well), press the system power button. Normally during cart shutdown, you hear a hiss, and a small amount of condensed water will discharge.
Computer Workstation
The workstation consists of a computer compatible with Microsoft Windows XP Professional operating system, a flat-screen monitor, and an optional printer.
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: a drawer a cover three optical sensors an electronics module a tube lifter a 40-tube carousel
For more information, see BD FACS Loader Option on page 33.
28
Figure 1-8 BD FACS Loader
29
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. The barcode reader reads most barcode standards, including Codabar, Code 128, Code 39 with checksum, and PDF417. It reads information from the BD FACS 7-color setup beads label into BD FACSCanto clinical software, and also reads coded patient information into a worklist.
Figure 1-9 Typical barcode reader (example)
ready light
trigger view from top
For information on installing and using the barcode reader with the BD FACSCanto, 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.
30
System Requirements
Software
Both included software packages must be installed: BD FACSCanto clinical software v2.1 Do not read FCS files created with v2.0 or v2.1 into previous versions of BD FACSCanto clinical software. Previous versions will show incorrect results. 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.
Workstation
BD FACSCanto workstation purchased through BD Biosciences
Compatible Tubes
12 x 75-mm polystyrene test tubes (BD Falcon tubes) 12 x 75-mm BD Trucount tubes BD FACS 7-color setup bead tubes
Bulk Fluids
BD FACSFlow solution BD FACSClean solution BD FACS shutdown solution
31
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
32
2
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
33
Loader Components
The Loader is mounted directly on the flow cytometer. The device consists of a drive system, tube lifter mechanism, spindle, and optical sensors, all of which are attached to a sliding drawer. A cover fits over the drawer to protect you from moving parts during operation. Table 2-1 provides a brief description of Loader components.
tube guide alignment guide pin tube optical sensor carousel optical sensor tube lifter drawer center spindle
Table 2-1 Loader components
tube guide alignment guide pin carousel optical sensor tube optical sensor
guides tubes during lifting to ensure a proper seal aligns the carousel and keeps it in place reads the carousel ID scans the carousel to verify tube locations and match the associated worklist, and verifies a tube is in place before activating the tube lifter a stainless steel rod that lifts the sample tube from the carousel to the sample injection tube (SIT)
tube lifter
34
Table 2-1 Loader components (continued)

drawer center spindle drive system holds the carousel; slides out for easy access the axis around which the carousel spins; insert the carousel onto the Loader here the motor that rotates the carousel (not visible from exterior)
Carousel
The carousel accommodates up to forty 12 x 75-mm tubes. Each carousel has a unique ID printed on top and on an optically read label inside.
Figure 2-1 Sample-Prep Ready carousel
spindle hole
alignment hole
The Loader is compatible with both the green-tinted carousels labeled SamplePrep 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
35
Not all manufactured 12 x 75-mm tubes have been checked for proper functionality on the Loader. BD Biosciences has validated only disposable, 12 x 75-mm BD Falcon polystyrene test tubes, BD Trucount tubes, and BD FACS 7-color setup bead tubes.
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.
1 Verify that the cytometer is set up for automatic loading.

The adapter lever should be in the lowest position, the tube guide should be forward, and the aspirator arm bar should be horizontal (Figure 2-3). For detailed instructions, see Changing to Automatic Loading on page 41.
Figure 2-3 Automatic loading setup
adapter lever
tube guide
aspirator arm bar
36
2 If needed, remove the carousel from the Loader:

Open the Loader cover. Pull out the Loader drawer. Place your thumb on the center spindle, and press down with your thumb while gently pulling up on the carousel handle with two fingers, as shown.
3 Vortex the sample tubes and place them uncapped in the carousel(s)
according to the worklist. 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. To prevent binding during loading, make sure label thickness per tube is less than three labels. Flatten labels completely before placing a tube in the carousel.
37
4 Install the first carousel to be acquired on the Loader.

Position the spindle hole in the handle over the center spindle of the Loader drawer. Rotate the carousel until the alignment guide pin fits into the small alignment hole at the top of the carousel. Press down firmly to seat the carousel.
Figure 2-4 Seating the carousel in the Loader
spindle handle
alignment pin
5 Close the Loader drawer completely, and install the Loader cover.
The Loader scans and positions the carousel at tube 1. 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
38
Changing Operational Modes

Performing a few simple steps allows you to change from automatic loading to manual loading. See the following sections for instructions. Changing to Manual Loading (on this page) Changing to Automatic Loading on page 41
Changing to Manual Loading

To change to manual mode, follow these steps. Handle all biological specimens and materials as if capable of transmitting infection. Dispose of waste using proper precautions and in accordance with local regulations. Wear suitable protective clothing, eyewear, and gloves.
Materials
personal protective equipment
Procedure
1 Remove the Loader cover. 2 Pull out the drawer. 3 Remove any carousel in the Loader.
39
4 Move the tube guide arm all the way back and away from you (in manual
mode, you do not need the tube guide arm).
automatic mode
manual mode
5 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
6 (Optional) Rotate the aspirator arm bar vertically.
automatic mode
manual mode
You are ready to load tubes manually.
40
Changing to Automatic Loading

To change from manual loading to automatic loading, follow these steps. Handle all biological specimens and materials as if capable of transmitting infection. Dispose of waste using proper precautions and in accordance with local regulations. Wear suitable protective clothing, eyewear, and gloves.
Materials
personal protective equipment
Procedure
1 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
41
2 Rotate the aspirator arm bar to the horizontal position until it reaches the
stop pin.
manual mode
automatic mode
3 Move the tube guide arm all the way forward.

The tube guide arm is needed only when using the Loader.
manual mode
automatic mode
4 Pull out the drawer.

You are ready to load tubes automatically.
42
Maintaining the Loader

This section explains the cleaning procedure you should follow to keep your Loader in good condition.
Cleaning External Surfaces

To keep the Loader free from salt buildup, wipe down all external surfaces that have been exposed to sheath fluid. All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease. Use universal precautions when cleaning cytometer surfaces. 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
43
Procedure
To avoid potential shock, always switch off the power and unplug the AC power cord before you begin cleaning.
1 Switch off the cytometer power and unplug the AC power cord from the
wall socket.
2 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.
3 Wet a fresh cloth with DI water and wipe all exposed surfaces to prevent
corrosion.
4 Wipe all exposed surfaces with a clean, dry cloth.
44
3
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
45
Installing and Using the Barcode Reader

To prevent laser injury, do not stare into the reader or aim it at another persons eyes while the trigger is depressed. 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: reading information from the BD FACS 7-color setup beads label into BD FACSCanto clinical software (2D barcode symbology) reading coded patient information from sample tube labels into a worklist (1D barcode symbology)
NOTICE The barcode reader supplied with the BD FACS Sample Prep Assistant II (SPA II) is a 1D reader that cannot read information from BD FACS 7-color setup beads. Use only the Opticon LG2 Imager 2D barcode reader for reading information from the BD FACS 7-color setup beads label into BD FACSCanto clinical software. NOTICE Contact BD Biosciences before changing default barcode settings.
46
1 Plug the barcode reader into the USB port on the back of your computer.
The reader turns on automatically.
2 Make sure the green light on top of the reader is illuminated:

green light
3 In BD FACSCanto clinical software, navigate to the fields for which

information can be scanned. Lot information for BD FACS 7-color setup beads is scanned into the Setup Lot Information dialog from the setup values label supplied with the setup bead kit (NOT from the lot number on the setup bead tubes). Patient information is scanned from sample labels into the worklist ID field.
4 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. Scan the barcodes exactly as they are provided. Tip To make it easier to use, an optional stand is available for the barcode reader. See Bar Code Reader Parts on page 129.
Chapter 3: Barcode Reader Option
47
5 Aim the barcode reader at the center of the barcode. The barcode reader
has two focal lengths, 4 and 9 inches: For reading BD FACS 7-color setup bead labels, hold the barcode reader 9 inches from the labels. For reading patient information from sample tube labels, hold the barcode reader 4 inches from the labels.
6 Press and hold the trigger on the barcode reader until you hear a beep.
If the reader does not beep, adjust your distance from the barcode while continuing to hold the trigger. Tip To obtain a reading, keep the bar code reader aimed at the center of the label. Do NOT sweep across the label.
7 Compare software field values with the setup beads or sample label.
Cleaning the Barcode Reader

To prevent laser injury, do not stare into the reader or aim it at another persons eyes while the trigger is depressed. For best performance, keep the front window of the barcode reader clean. Do not touch the window directly, and wipe it only with a soft, non-abrasive cloth moistened with one of the following: isopropyl alcohol ethyl alcohol (denatured grade) Do not use BD FACSClean solution (or bleach) to clean or disinfect the barcode reader.
48
Barcode Symbologies
Although data entry using barcodes is generally more reliable than manual data entry, it is not guaranteed to be 100% accurate. To increase accuracy when using the barcode reader, enabling checksums is recommended. Using barcode symbologies without checksums increases the likelihood of incorrect information transfer, including sample ID assignments. This can result in a mismatch of sample IDs and sample results. 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 Code 128 Code 39 Recommendation Preferred. Acceptable if barcode labels are printed with the checksum digit. By default, the barcode reader recognizes the checksum digit when reading the Code 39 symbology. However, if labels are printed without a checksum digit, contact your BD service representative for instructions on disabling the checksum feature. The barcode reader does not support the checksum feature when reading the Codabar symbology.
Codabar
Chapter 3: Barcode Reader Option
49
2D Barcode Symbologies
BD Biosciences has evaluated 2D barcode symbology to read the target values of BD FACS 7-color setup beads when using BD FACSCanto clinical software. 2D barcode symbology is required to read all target values with one scan. 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.
50
4
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
51
Scheduled Maintenance
For optimal cytometer functioning, perform the following maintenance according to the recommended schedule.
Procedure Fluidics startup Description and Location Replaces BD FACS shutdown solution with BD FACSFlow solution Refer to the BD FACSCanto Instructions for Use. Fluidics shutdown Replaces BD FACSFlow solution with BD FACS shutdown solution Refer to the BD FACSCanto Instructions for Use. Wipe down SIT and aspirator arm Prevents saline deposit buildup Refer to the BD FACSCanto Instructions for Use. See Emptying the Waste Container on page 54. Removes air from fluid filters, ensuring they will not dry out See Purging the Fluidics Filters on page 57. Change the waste tank cap See step 6 on page 56 (in Emptying the Waste Container procedure). Monthly Daily and as needed Weekly Daily Daily Frequency Daily
Empty the waste Purge fluid filters
52
Procedure Decontaminate fluidics (Long Clean)
Description and Location Decontaminates the internal sheath path with BD FACSClean solution, then rinses with BD FACS shutdown solution See Decontaminating the Fluidics System (Long Clean) on page 59.
Frequency Monthly and before service calls
Replace the air filter
Ensures proper cytometer performance See Replacing the Air Filter on page 60.
Every 6 months
Replace fluid filters
Keeps fluids free of particulates See Replacing Fluidics Filters on page 63.
Every 6 months or when increased debris observed in FSC vs SSC plots Every 6 months
Schedule preventative maintenance by BD service
Chapter 4: Maintenance
53
Emptying the Waste Container

All biological specimens and materials coming into contact with them can transmit potentially fatal disease. To prevent exposure to biohazardous agents, expose waste container contents to bleach (10% of total volume) before disposal. 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
1 Ensure the cytometer is not acquiring events.
54
2 Detach the waste containers sensor and fluid line.

Pull the sensor straight out. Press the metal clip on the fluid line.
sensor
waste fluid line with metal clip
The waste tank can become pressurized when the cytometer is running. Always disconnect the tank from the fluidics cart before you empty it. Wait at least 30 seconds for pressure to dissipate before you remove the waste cap or level sensor cap.
3 Remove the disposable waste cap and attached trap from the container.
Place the assembly on the bench label-side up. Do not wet the cap. If you see liquid inside the trap, remove the drain plug and fully drain the liquid before you replace the plug.
trap
drain plug
55
4 Empty the bleach-exposed waste. 5 Add approximately 1 L of bleach to the empty waste container (10 L
container).
6 If one month has passed since the last cap change:

Detach the cap from the trap. Replace it with a new cap. Write the date on the cap label.
Waste (A)
space for date

338677 Rev A
To prevent tank overpressurization, do not overtighten the trap or attached filter cap. Tighten each component only until it is hand-tight. Do not use sealants such as Teflon tape or other adhesives.
7 Screw the cap assembly onto the tank and hand-tighten until it is fully
closed.
8 Re-attach the sensor and fluid lines.
56
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
paper towels proper protective equipment
Procedure
1 Run the Prime After Tank Refill for all fluids to ensure fluid lines are
primed. See Priming Fluidics Lines on page 71.
2 Place a few paper towels beneath the filter to be purged to absorb any fluid
leakage.
57
3 Loosen the bleeder valve near the top of the filter by turning it
counterclockwise. NOTICE Tip 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
4 Wait until fluid seeps out.

Fluid should seep from the open valve within 30 seconds. If no fluid appears, make sure the corresponding cubitainer is not empty or detached from the cart. 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.
5 Close the valve by turning it clockwise. 6 Repeat steps 2 through 5 with the remaining filters.
58
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. NOTICE This procedure takes 75 minutes to complete.
Materials
undiluted bleach (for waste tank) BD FACSClean solution (approximately 275 mL) BD FACS shutdown solution (approximately 1,100 mL)
Procedure
1 Check all fluid levels; empty the waste if needed. 2 Select Cytometer > Cleaning Modes > Long Clean.
A confirmation dialog displays.
3 Click OK to continue. Once you have begun the Long Clean, you cannot
abort the process. A completion message displays when the cleaning cycle finishes.
Figure 4-1 Long Clean completion message
BD FACSCanto clinical software
BD FACSDiva software
59
4 Click OK.
If the completion message does not display after 90 minutes, verify there are no error messages in the Status tab of the Cytometer window. If the cleaning mode fails, see Fluidics Cart Troubleshooting on page 101.
5 Select to shut down or continue.

To shut down, select Cytometer > Fluidics Shutdown. Repeat Fluidics Shutdown twice more. Quit the software and turn off the power to the cytometer. To continue, select Cytometer > Fluidics Startup. Repeat Fluidics Startup twice more. BD FACS shutdown solution can lyse cells. The Fluidics Startup procedure removes BD FACS shutdown solution from the interior reservoir and fluid lines and replaces it with BD FACSFlow solution. Tip 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
Replacement filter (see Other Replacement Parts on page 129 for ordering)
Procedure
1 Turn off the cytometer power. 2 Open the side door.

Press the black button; a handle will pop out.
60
Twist the handle and pull.
3 On the doors interior, turn the pegs along the upper edge of the filter and
remove the old filter.
61
Dispose of the old filter. It cannot be reused.
4 Install a new filter, making sure the arrow on top of the filter points in
toward the cytometer (arrow indicates direction of air flow).
5 Turn the pegs to hold the filter in place. 6 Close the side door:
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.
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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 felt tip pen
63
Procedure
1 Place a few paper towels beneath the filter to collect drips. 2 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
3 Position the new filter and connect the couplings.

Tip Write todays date on the filter so you will know when to replace it.
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4 Loosen the top bleeder valve by turning it counterclockwise.

NOTICE Tip Do not loosen the bottom bleeder valve. Ensure it is tightened.
Do not completely unscrew the valve. If you do, it will come off.
5 Wait until fluid seeps out.

Fluid should seep from the open valve within 30 seconds. If no fluid appears, ensure the corresponding cubitainer is not empty or detached from the cart. 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.
6 Close the valve by turning it clockwise.

Repeat the procedure for other fluid filters to be replaced.
65
Unscheduled Maintenance
Perform these maintenance procedures as needed.
Procedure Replace cubitainers Prime fluid lines Description and Location See Changing a Cubitainer on page 68. Removes air from fluid lines See Priming Fluidics Lines on page 71. Remove an air lock Removes air from filter and restores flow of fluid See Removing an Air Lock on page 72. Clean external surfaces Keeps surfaces free from saline deposit buildup after exposure to sheath fluid See Cleaning External Surfaces on page 75. De-gas Flow Cell Removes bubbles from the flow cell See Removing Bubbles from the Flow Cell on page 76. Clean Flow Cell Runs BD FACSClean solution through the SIT and flow cell See Cleaning the Flow Cell on page 77. Bubble Filter Purge Removes air from the bubble filter See Purging the Bubble Filter on page 78. When poor optical performance indicates additional cleaning is needed If fluidics run dry, or when CVs are poor As needed during daily startup Frequency As needed Whenever a fluidics line is disconnected to change a cubitainer or perform other maintenance When fluidics are not functioning properly (flow cell will not fill, or there is backflush into sample tube) As needed
66
Procedure Decontaminate the fluidics system for storage
Description and Location Cleans out the fluidics lines with BD FACSClean solution, then fills them with BD FACS shutdown solution See Decontaminating the Fluidics System for Storage on page 80.
Frequency Before long-term storage
Replace the Bal seal
Replaces worn Bal seal See Replacing the Bal Seal on page 80.
When the seal becomes worn or cracked (sample tubes will not pressurize) After the circuit breaker is tripped When a cable is disconnected As needed When instructed to by a BD Biosciences service representative When cart will not function because of power surge or other electrical event
Reset the cytometer circuit breaker Reattach Ethernet and RS232 cables Reconnect fluidics cart tubing Replace fluidics level sensors
See Resetting the Cytometer Circuit Breaker on page 84. See Reconnecting the Ethernet and RS232 Cables on page 85. See Reconnecting the Fluidics Cart Tubing on page 85. See Replacing the Fluidics Level Sensors on page 88.
Replace fluidics cart fuses See Replacing the Fluidics Cart Fuses on page 91.
67
Changing a Cubitainer
Three fluidics cubitainers (disposable boxes of approved fluids) and a waste container fit onto the cart in the following configuration:
BD FACS shutdown solution BD FACSClean solution
waste container
BD FACSFlow solution
Each cubitainer and the waste tank has its own color-coded port. Connect containers to the following ports:
Container waste BD FACSFlow solution BD FACS shutdown solution BD FACSClean solution Port Label Waste (A) BD FACSFlow Port Color orange-red blue
BD FACS Shutdown Solution green BD FACS Cleaning Solution green
68
Procedure
1 Ensure the cytometer is not acquiring events. 2 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.
3 Unscrew the cap on the cubitainer.
69
4 Remove the cap and sensor assembly and set it aside.
5 Put a new cubitainer onto the fluidics cart. 6 Replace the cap assembly and hand-tighten it until it is fully closed. 7 Reattach the sensor line and fluid line to the cart.
To attach the sensor line, gently rotate until the connection aligns, and then push. To attach the fluid line, push the coupling into the port until it clicks.
To ensure that the appropriate solutions are dispensed, match the label on the container to the port on the fluidics cart.
8 Prime the fluidics.

Important: Continue with the procedure in Priming Fluidics Lines on page 71.
70
Priming Fluidics Lines

Use the Prime After Tank Refill command to remove air from the fluid lines after you change a cubitainer or detach the fluidics lines for other maintenance.
1 Select Cytometer > Cleaning Modes > Prime After Tank Refill. 2 Select the checkboxes for the cubitainers you changed; click OK.
3 Click OK when the completion message displays.
71
Removing an Air Lock

If too much air gets into the sheath filter, it becomes impermeable to fluid and an air lock can develop. Although it is most likely to occur for the sheath filter, an air lock can develop in any of the fluidics filters. All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease. Use universal precautions when cleaning cytometer surfaces. Wear suitable protective clothing and gloves.
Materials
paper towels bypass tubing
Procedure
1 Place a few paper towels beneath the air-locked filter to collect drips. 2 Remove the filter by pressing the metal tabs.
Figure 4-3 Removing a filter
metal tab
metal tab
72
3 Install bypass tubing in place of the filter.

Push the tubing into each quick-disconnect port until you hear a click.
bypass tubing, installed
4 Select Cytometer > Cleaning Modes > Prime After Tank Refill. 5 Select the checkbox that corresponds to the filter you have bypassed. 6 Click OK.
7 When the prime finishes, remove the bypass tubing. 8 Reattach the filter to the fluidics cart. 9 Repeat the Prime After Tank Refill command.
73
10 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
74
Cleaning External Surfaces

To prevent salt buildup, wipe down all external cytometer surfaces that have been exposed to sheath fluid. NOTICE Over time, saline deposits may develop on the interior surface of the SIT Bal seal retainer, causing an incomplete SIT flush between samples. If this occurs, you might need to remove and clean the retainer with DI water. See Figure 4-5 on page 80 and steps 4, 7, and 8 of the Replacing the Bal Seal procedure that starts on page 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. Use universal precautions when cleaning cytometer surfaces. Wear suitable protective clothing and gloves. To prevent damage, do not use isopropyl alcohol or ethanol on any cytometer or fluidics cart surface.
Materials
BD FACSClean solution DI water clean, lint-free cloths or disposable wipe
75
Procedure
1 Switch off the cytometer power and unplug the AC power cord.
To avoid potential shock, always switch off the power and unplug the AC power cord before you begin cleaning.
2 Wipe all accessible surfaces with BD FACSClean solution. 3 Wet a fresh cloth with DI water and wipe all bleach-exposed surfaces to
prevent corrosion.
4 Wipe all exposed surfaces with a clean, dry cloth.
Removing Bubbles from the Flow Cell

Use the De-gas Flow Cell command to remove bubbles from the flow cell.
1 Select Cytometer > Cleaning Modes > De-gas Flow Cell. 2 Click OK when the completion message displays.
3 Check the flow cell for bubbles.

If you still see bubbles, repeat the process.
76
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. Perform this procedure when poor optical performance indicates that additional cleaning is needed.
Materials
3 mL of BD FACSClean solution one 12 x 75-mm polystyrene BD Falcon tube
Procedure
1 Select Cytometer > Cleaning Modes > Clean Flow Cell. 2 If you have a Loader, remove the carousel. 3 When prompted, manually install a tube containing approximately 3 mL of
BD FACSClean solution onto the SIT, and click OK.
77
A progress message displays during the cleaning.
4 After the completion message displays, wait 5 minutes.

Waiting allows the BD FACSClean solution to dissolve deposits in the flow cell cuvette.
5 Click OK. 6 Remove the tube from the SIT. 7 Clear BD FACSClean solution from the flow cell and fluidics lines:
Before running setup or samples, run Fluidics Startup. If you are shutting down without running more samples, run Fluidics Shutdown.
Purging the Bubble Filter

The bubble filter sits between the fluidics tanks and reservoirs and the flow cell and ensures that the flow cell remains bubble-free. However, should the fluidics run dry, you might need to remove air from the bubble filter. Poor CVs might indicate a need to purge the bubble filter.
1 Select Cytometer > Cleaning Modes > Bubble Filter Purge.
78
2 A dialog indicates Bubble Filter Purge progress. Wait until the purge
finishes.
3 Click OK when the completion message is displayed.
4 Repeat steps 1 through 3 until bubble-free liquid enters the flow cell from
the bubble filter.
5 When finished, select Cytometer > Cleaning Modes > De-gas Flow Cell.
79
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.
1 Perform steps 1 through 4 in Decontaminating the Fluidics System (Long

Clean) on page 59.
2 Run fluidics shutdown. 3 Shut down the software and turn off the power to the cytometer.
Replacing the Bal Seal

The Bal seal is a Teflon ring that allows the sample tube to pressurize. Over time, this seal becomes worn or cracked and requires replacement. Replace the seal when persistent sample tube pressure errors occur. Over time, saline deposits may develop on the interior surface of the SIT Bal seal retainer, causing an incomplete SIT flush between samples. Therefore, you should always rinse the retainer in DI water when you replace a Bal seal.
Figure 4-5 Bal seal and retainer
Bal seal SIT retainer
80
Materials
proper protective equipment replacement Bal seal DI water
Procedure
1 Turn off the cytometer. 2 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). If you do not change the cytometers mode, you will not be able to access the Bal seal.
81
3 Move the aspirator arm to the left and hold it.
aspirator arm bar aspirator arm
4 Remove the retainer from the SIT by turning it in the direction shown.
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5 Remove the Bal seal by pulling it off with your fingers.
Bal seal
6 Install the new Bal seal

spring-side up.
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7 Rinse the retainer in DI water to remove saline deposits before replacing it

on the SIT.
8 Reinstall the retainer over the SIT and tighten in the direction shown.
9 Test by manually loading a tube onto the SIT and running fluid.
Resetting the Cytometer Circuit Breaker

1 Toggle the cytometer circuit breaker switch to on.
main power button
cytometer circuit breaker, on
2 If it is off, turn on the cytometer main power.
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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. As the make and model of the workstation might vary, refer to the documentation that came with your system for information.
Reconnecting the Fluidics Cart Tubing

Should any plugs, cords, or tubings become accidentally disconnected, use the following diagrams to reconnect the fluidics cart to the cytometer (Figure 4-7 on page 86). Tip The ports and connectors are color coded.
85
Figure 4-7 Cytometer and fluidics cart ports
h i
d k o
g cytometer ports fluidics cart ports
Cytometer ports and their corresponding fluidics cart ports are listed in Table 4-1. For example, ensure the tubing for f. Sheath (B) connects to the port labeled i. Fluid Out. Table 4-2 on page 87 lists port functions.
Table 4-1 Correspondence of cytometer ports to fluidics cart ports
Port or Button on Cytometer a. System Power b. Power Out c. Air In d. Waste (A) e. Communications f. Sheath (B) g. Waste (A) Port on Fluidics Cart k. Power In m. Air Out o. Waste j. Communication i. Fluid Out n. Waste h. On/Off l. Air In
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Do not plug the fluidics cart power cord into a wall outlet. Plug the cord into the cytometer only. This ensures proper electrical grounding and protects against electrical shock or damage to the cytometer.
Table 4-2 Function of ports, buttons, and switches
Port or Switch a. System Power b. Power Out c. Air In d. Waste (A) e. Communications f. Sheath (B) g. Waste (A) h. On/Off i. Fluid Out j. Communication k. Power In l. Auxiliary Air In m. Air Out n. Waste o. Waste Additional Information Powers both cytometer and fluidics cart Connects to fluidics cart 705 psi Waste out (aspirated) Data port BD FACSFlow solution port Waste out (non-aspirated) Auxiliary air supply switch. Keep in off position unless connected to house air. BD FACSFlow solution port Data port Connects to cytometer. Do not connect to wall outlet There will be no tubing on this port unless connected to house air. Sends compressed air to cytometer Waste in (non-aspirated) Waste in (aspirated)
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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. All biological specimens, and materials coming into contact with them, can transmit potentially fatal disease. The waste tank can become pressurized when the cytometer is running. Always disconnect the tank from the fluidics cart and wait at least 30 seconds for pressure to dissipate before you remove the level sensor cap.
Materials
replacement fluidics sensor probesee Other Replacement Parts on page 129 for ordering proper protective equipment
88
Procedure
1 Ensure that the cytometer is not acquiring events. 2 Detach the sensor and fluid line from the cart. 3 Unscrew the cap on the cubitainer. 4 Remove the level sensor assembly and discard into a suitable receptacle.
89
5 Put a new level sensor assembly on the cubitainer or tank. Hand-tighten the
cap until it is fully closed.
6 Ensure use of the correct assembly (Figure 4-8).

Figure 4-8 Level sensor assemblies
waste level sensor (red connector)
BD FACSFlow level sensor (blue connector)
BD FACSClean solution and BD FACS shutdown solution level sensor (green connector)
7 Reattach the sensor line and fluidics lines.

To ensure that the appropriate solutions are dispensed, make sure the label on the container matches the labeled port on the fluidics cart.
8 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.
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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.
1 Shut down the cytometer and turn off the cytometer power. 2 Unplug the power cord from the wall outlet and the fluidics cart.
Removing the plug allows easier access to the fuse door.
3 Insert a small screwdriver into the slot and gently pry the fuse door open.
slot for screwdriver fuse access door circuit breaker empty socket, plug removed
91
4 Gently pry out the fuse drawer until you can grip it.
fuse drawer
fuse door
5 Remove the fuse drawer, and remove and dispose of the old fuses.
Tip Note the positions of the old fuses before you remove them. Duplicate these positions with the new fuses.
6 Replace both fuses.

Make sure the fuse sits as shown:
right
wrong
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For protection against risk of fire, replace fuses only with those provided by BD Biosciences.
7 Make sure the text for your areas voltage is right-side up.
8 Slide the drawer back into the cytometer until it snaps into place. 9 Close the fuse access door. 10 Reconnect the power cord to the fluidics cart. 11 Plug the cytometer power cord into the wall outlet and switch on the power.
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94
5
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. If you need additional assistance, contact BD Biosciences. Refer to our website, bdbiosciences.com, for up-to-date contact information. Troubleshooting suggestions can be found under the following topics: Cytometer Troubleshooting on page 96 Fluidics Cart Troubleshooting on page 101 Loader Troubleshooting on page 102
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Cytometer Troubleshooting
Observation Flow cell will not fill Possible Causes Air in bubble filter Recommended Solutions Run the Bubble Filter Purge command at least once (you might need to run it several times). When finished, run De-gas Flow Cell. See Purging the Bubble Filter on page 78. Turn on the power to the fluidics cart by resetting the fluidics cart circuit breaker switch (Figure 1-7 on page 27). Always use the cytometer power button, located on the left side of the cytometer, to turn the system off and on. Check air tubing connections. Check air tubing for kinks. Sheath line disconnected
Fluidics cart power off
No pressure in the plenum
1 Check cubitainer-to-fluidics
cart, and fluidics carttocytometer connections. See Reconnecting the Fluidics Cart Tubing on page 85.
2 Check tubing for kinks.

BD FACSFlow cubitainer empty Air in BD FACSFlow filter (fluidics cart) Air lock in BD FACSFlow filter (on fluidics cart) Replace the BD FACSFlow cubitainer. See Changing a Cubitainer on page 68. Purge air from the filter. See Purging the Fluidics Filters on page 57. See Removing an Air Lock on page 72.
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Cytometer Troubleshooting (continued)

Observation Flow cell will not fill (continued) Possible Causes No air pressure in cart Recommended Solutions If the cart is attached to an auxiliary air supply, switch on auxiliary air. If the cart is not attached to an auxiliary air supply, switch off auxiliary air. Fluidics pressure errors Air lock in filter Check the filter in the fluidics cart. Verify the bottom bleeder valve on the filter is fully tightened. Open the top bleeder valve. If no fluid leaks out, remove the air lock as described in Removing an Air Lock on page 72. Transfer sample to new tube. Make sure you are using appropriate tubes. See System Requirements on page 31. Bal seal improperly installed or worn Air lock in BD FACSFlow filter Liquid leakage around cytometer base Interior valve failure Reinstall or replace the Bal seal. See Replacing the Bal Seal on page 80. See Removing an Air Lock on page 72.
Fluid backfill into sample tube
Cracked tube
1 Turn off the cytometer power. 2 Clean up the liquid, using

proper precautions.
3 Call BD Biosciences.
QC fails after Long Clean Residual BD FACSClean solution in lines Run Fluidics Startup to flush the system with sheath fluid. Repeat until QC passes.
Chapter 5: Troubleshooting
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Observation Cytometer on, but no response to software commands Possible Causes Bad keyboard or mouse connection Recommended Solutions Check keyboard or mouse connections to computer. Refer to the documentation that came with your workstation.
Communication failure (Ethernet error) between computer and cytometer
1 Turn off the computer and the

cytometer.
2 Reseat the Ethernet cable,

located on the right side of the flow cytometer. See Figure 4-6 on page 85.
3 Turn on the cytometer, then the

computer.
4 Using hyperterminal, type

reboot. Verify that VxWorks launches and no errors are reported. Software does not connect to cytometer BD FACSDiva software running at same time as BD FACSCanto clinical software Cytometer power is off Internal firmware error Cytometer and fluidics cart will not turn on Power cords disconnected from wall socket or cytometer Cytometer circuit breaker tripped Blown fuse in cytometer controller Quit the software you are not using or put the software in Standby. Turn power on. Cycle power on cytometer. Reconnect the power cord to the wall socket or the cytometer. Switch on the cytometer circuit breaker, followed by the cytometer power (if off). Turn off the cytometer main power and call BD Biosciences.
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Observation Tube does not load, or sample tube does not fit snugly on SIT Possible Causes Improper sample tube used Recommended Solutions Use Falcon brand 12 x 75-mm polystyrene test tubes, BD Trucount tubes, or BD FACS 7-color setup bead tubes. See System Requirements on page 31. Reinstall or replace the Bal seal. See Replacing the Bal Seal on page 80. Transfer sample to new tube. Make sure you are using appropriate tubes. See System Requirements on page 31. Bal seal improperly installed or worn Adapter lever in incorrect position for manual loading Reinstall or replace the Bal seal. See Replacing the Bal Seal on page 80. Make sure the lever is in the manual loading position. Modify the cytometer appropriately. See Changing to Manual Loading on page 39. Set G0/G1 diploid population peak at channel 25,000 or less.
Bal seal improperly installed or worn Defective or cracked tube
Higher than expected CVs for some (brightest) populations during DNA analysis >20% difference between Measured and Reference laser power BD FACSDiva software not launching
Possible pre-amp nonlinearity with very bright signals Flow cell access door open
Close the door completely.
Cytometer.dat file not found
Install saved cytometer.dat file, or call BD Biosciences for assistance.
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Observation Fluid leaking from SIT or aspirator arm Possible Causes Remove tube sequence not followed correctly, cytometer still in acquisition mode Interior valve failure or leak Recommended Solutions Perform the tube removal sequence again, even if a tube is no longer on the SIT.

proper precautions.
3 Call BD Biosciences.
Waste line to fluidics cart disconnected

proper precautions.
3 Check that waste lines are

securely plugged in.
4 Turn on the cytometer power.
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Fluidics Cart Troubleshooting

Observation Water leakage around fluidics cart base Possible Causes Normal condensation overflow from pressure relief valve Extremely humid climate Bleeder valve on fluidics cart filter open Recommended Solutions
1 Turn off the cytometer power. 2 Clean up the water.
1 Turn off the cytometer power. 2 Clean up the liquid. 3 Check and close all bleeder
valves. See Purging the Fluidics Filters on page 57.
Broken fluid line Fluidics cart will not power on, cytometer on Cart circuit breaker off Auxiliary air supply on, cart not normally connected to auxiliary air Power cord from fluidics cart to cytometer disconnected Fuse blown
Contact BD Biosciences. Switch on the cart circuit breaker. Toggle the auxiliary air supply off. When auxiliary air is on, the carts own air pumps turn off. Connect both ends of the cord. Replace the fluidics cart fuses. See Replacing the Fluidics Cart Fuses on page 91.
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Loader Troubleshooting
Observation Tube does not load Possible Causes Loader not set up for automatic loading Recommended Solutions Set up the Loader according to Changing to Automatic Loading on page 41. Make sure both the adapter lever and tube guide were moved as shown. Make sure the Loader drawer is completely shut. Make sure the tube is free of bulky labels or tape. Tube lifter hitting carousel during ascent Make sure the carousel is properly engaged with the alignment guide pin. If the problem persists, contact BD Biosciences for assistance. Contact BD Biosciences. Clean the tube optical sensor (outer sensor), as described in Cleaning External Surfaces on page 43. Wipe tube. Turn tube. Set a stopping time in BD FACSDiva software. Refer to the BD FACSCanto Instructions for Use. Degas flow cell. See Removing Bubbles from the Flow Cell on page 76.
Tube not completely raised
Tube lifter failure Tube missed, or missing tube messages in software Salt crystal buildup on optical sensors
Condensation on tube Tube label reflection Tube runs dry, Loader not advancing to next sample Dilute sample or rare events
Bubble in flow cell diverts stream
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Loader Troubleshooting (continued)

Observation Tube stuck on SIT, software not responding Possible Causes Loader not set up for automatic loading Software problem Recommended Solutions Manually remove the tube from the SIT.
1 Hold the tube and move the

aspirator arm aside.
2 Gently pull the tube off the SIT

and replace in carousel.
3 Release the aspirator arm. 4 Restart the worklist run. 5 If needed, modify the cytometer
for automatic loading. See Changing to Automatic Loading on page 41. Carousel not rotating correctly Carousel not engaged with alignment pin on drawer Rotate the carousel on the spindle until the alignment guide pin engages with the alignment hole, and press down. See step 4 on page 38. If the problem persists, contact BD Biosciences for assistance. Rotate the bar horizontally. See Changing to Automatic Loading on page 41.
Aspirator arm bar vertical
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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
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Flow Cytometry
Flow cytometry measures certain properties of particles, such as size and internal complexity, using light. To do this, a flow cytometer needs a method to move the particles past a light source. It also needs a way to collect and convert the light scattered or emitted by the particles into electrical signals. Most modern cytometers use lasers as the light source and transport the particles under investigation past the lasers using fluid or air. The major systems in a cytometer that moves particles by fluid transport include a fluidics system an optical system an electronics system
Fluidics System
A fluidics system in a flow cytometer moves particles in fluid through a flow cell, past a laser beam, and then into a waste tank.
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-m filter to a pressurized interior reservoir within the cytometer, called the plenum. The plenum maintains a nearly constant fluid level and dampens pump pulsation. As a result, sheath pressure does not vary with the level of fluid in the sheath cubitainer. The plenum delivers sheath fluid that has been filtered for air via a bubble filter to the flow cell with minimal flow rate variations. Figure A-1 on page 107 shows the fluidics pathway for the BD FACSCanto.
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Figure A-1 Sheath and sample flow pathways for the BD FACSCanto cytometer
Sheath Flow sheath tank test tube sheath filter interior reservoir (plenum) bubble filter Sample Flow
sample injection tube (SIT)
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. In BD FACSDiva software select Cytometer > Cytometer Status Report.
Appendix A: Technical Overview
107
Tube to Flow Cell

When you place a tube on the cytometer, sample travels up the sample injection tube (SIT) in a separate, pressurized stream. It arrives in the lower chamber of the flow cell at a slightly higher pressure relative to the sheath fluid. The conical shape of the lower chamber creates a hydrodynamic focusing that carries the sample particles in a stable stream upward through the center of the flow cell. In the flow cell, laser beams interrogate one sample particle at a time. (Figure A-2). The difference in pressure between the sample stream and sheath fluid stream can be used to vary the diameter of the sample stream, also known as the sample core. Increasing the sample pressure increases the core diameter and therefore the flow rate (Figure A-2). 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.
108
Figure A-2 Hydrodynamic focusing of the sample core through the flow cell
low sample pressure (10 L/min)
red beam blue beam
high sample pressure (120 L/min)
sheath fluid sample
sheath fluid
sheath fluid sample
sheath fluid
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. Because the laser beam is focused and particles move rapidly through the flow cell, the scatter or fluorescence emission has a very brief durationonly a few microseconds. This brief flash of light is collected, filtered, and then converted into an electrical signal by the detectors.
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Figure A-3 Light pathway through the BD FACSCanto flow cytometer

stained particle focusing lens
fiber optic cables
prisms
collecting lens
forward scatter diode detector
steering plate flow cell red laser
obscuration bar LP mirror
PMT detector
fiber optic cable blue laser
BP filter
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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). FSC and SSC intensities indicate physical properties of cells: FSCindicates relative differences in the size of the cells or particles SSCindicates relative differences in the internal complexity or granularity of the cells or particles
Figure A-4 Forward scatter (FSC) and side scatter (SSC)

side scatter
laser
forward scatter
111
Fluorescence
When cells or particles stained with fluorochrome-conjugated antibodies or other dyes pass through a laser beam, the dyes can absorb photons (energy) and be promoted to an excited electronic state. In returning to their ground state, the dyes release energy, most of which is emitted as light. This light emission is known as fluorescence. Fluorescence is always a longer wavelength (lower-energy photon) than the excitation wavelength. Some fluorescent compounds emit at a much longer wavelength than their excitation wavelength. PerCP absorbs blue light (488 nm) and emits red light (675 nm); other fluorochromes, such as FITC, absorb blue light (488 nm) and emit green light (530 nm). These differences between excitation and emission allow one laser to excite many fluorochromes. The emission spectra for some commonly used fluorochromes are shown in Figure A-5.
Figure A-5 Emission spectra of commonly used fluorochromes
100%
normalized intensity
FITC
PE
APC
PerCP-Cy5.5
PE-Cy7 APC-Cy7
PE-Cy7
APC-Cy7
0% 400 500 600 wavelength (nm) 700 800
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Optical Filters
Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors. When photons encounter an optical filter, they are either transmitted, absorbed, or reflected.
Figure A-6 Effect of an optical filter on incident photons
photons absorbed photons transmitted photons reflected
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, a 500-LP filter permits wavelengths longer than 500 nm to pass through it and either absorbs or reflects wavelengths shorter than 500 nm (Figure A-7 on page 114).
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Figure A-7 Longpass filter

longpass
% transmission
wavelength (nm)
Not all light with shorter wavelengths is absorbed or reflected. Some will still pass through.
Bandpass Filter
A BP filter transmits a relatively narrow range or band of light. Bandpass filters are typically designated by two numbers. The first number indicates the center wavelength and the second refers to the width of the band of light that is passed. 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.
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Figure A-8 Bandpass filter

bandpass
% transmission
wavelength (nm)
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). The more sensitive PMTs are used to detect the weaker signals generated by SSC and all fluorescence channels. In BD FACSCanto clinical software, the fluorochromes are preset. In BD FACSDiva software, you can use the Cytometer Configuration window to specify the fluorochrome that will be measured at each PMT detector. You can also add additional parameters to your configuration and choose the appropriate fluorochrome within your software experiment.
Detector Arrays
On BD FACSCanto flow cytometers, PMTs are organized onto an octagon and a trigon. The octagon has five PMTs. The trigon has two PMTs. These arrays
115
efficiently direct the emitted light from each fluorochrome to a specific PMT, through placement of LP mirrors and BP filters. When the collected light leaves the fiber optic cable at the octagon, it first meets a 735 LP mirror (Figure A-9 on page 116). The mirror allows light with wavelengths greater than 735 nm to pass and reflects light with lower wavelengths to the next PMT.
Figure A-9 Light pathway around an octagon
A
78
750810 nm
515545 nm
0/6
564606 nm
73 5L
530/30
502LP
585/4 2
556LP
655
LP
LP
488/1
67
> 670 nm
483493 nm
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.
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Figure A-10 Excitation and emission spectra of PE-Cy7

blue laser light excitation bandpass 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. Light from dyes such as PerCP-Cy5.5 pass through this filter. The beam continues around the array, with different light spectrum ranges collected at successive PMTs Side scatter signals (blue laser light that was deflected by the internal irregularities of a particle) are collected at PMT E.
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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.
Figure A-11 Spillover of FITC into PE detector
BP filters
normalized intensity
emission spectrum of FITC emission spectrum of PE
FITC spillover into PE
For example, FITC appears primarily in the FITC detector, but some of its fluorescence spills over into the PE detector. PE appears primarily in the PE detector, but some of its fluorescence spills over into the FITC detector. This spillover must be corrected (or compensated for). Figure A-11 shows that a portion of the FITC emission will be detected by both the FITC and PE channels. This can be seen in an x, y plot of FITC vs PE (Figure A-12 on page 119).
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The green circle represents a population stained with FITC; the gray circle represents a population negative for both FITC and PE.
Figure A-12 Theoretical display of FITC vs PE before compensation
PE
FITC
If the fluorescence is to be assigned to PE, the FITC signal must be removed from the PE channel, as indicated by the arrow. Both PE and FITC fluoresce in the yellow (575 nm) range, so there is no way to isolate the emission from each fluorochrome optically. Instead, fluorescence compensation moves the FITC population out of the PE positive area (Figure A-13).
Figure A-13 FITC signal properly compensated out of the PE channel
PE
FITC
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.
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Once compensation has been set for one sample, the spectral overlap setting (compensation value) remains valid whether a dim or bright sample is run. Figure A-14 illustrates this principle. Although the signals differ in intensity, the percentage of signal detected in the FITC and PE channels remains constant.
Figure A-14 Two FITC signals of different intensity
FITC PE
different intensity FITC signals normalized intensity
same proportion or percentage of spillover in PE channel
Electronics System
Any discussion of digital electronics requires a basic understanding of the bit. Computers and digital circuits use bits (binary numbers consisting of ones and zeros) to pass information. A 4-bit number has 4 binary digits that are either 1 or 0. A 10-bit number has 10 binary digits that are either 1 or 0. An 18-bit number has 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.
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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
signal intensity time signal intensity time signal intensity time laser beam a particle
A pulse begins when a particle enters the laser beam. At this point, both the beam intensity and signal intensity are low.
b
The pulse reaches a maximum intensity or height when the particle reaches the middle of the beam, where the beam and signal intensity are the brightest. The peak intensity, or height of the pulse, is measured at this point.
c
As the particle leaves the beam, the pulse trails off.
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Pulse Measurements
The pulse of electricity travels from the PMTs to the electronics boards within the cytometer.
Figure A-16 Electronics boards
PMT PMT PMT PMT PMT PMT PMT FSC diode
preamp
preamp
preamp
preamp
preamp
preamp
preamp
preamp
ADC
ADC
ADC
ADC
ADC
ADC
ADC
ADC
RAM
RAM
RAM
RAM
RAM
RAM
RAM
RAM
FPGA
FPGA
FPGA
FPGA
FPGA
FPGA
FPGA
FPGA
digital signal processor
acquisition board
digital signal processor
acquisition board
master board
embedded computer
workstation
The pulse is amplified and then sent to a 14-bit analog-to-digital converter (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).
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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). After the numbers pass through the digital delay and are aligned, the numbers are then sent to a field programmable gate array (FPGA) for further processing. During this process, when a pulse exceeds the user-assigned threshold, its height and area are simultaneously calculated by the FPGA.
Figure A-18 Pulse measurements
signal intensity
pulse height
pulse area
time
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An FPGA calculates pulse height and area in the following manner: The maximum digitized value of all data points for the pulse becomes the pulse height. The sum of all data points that occur within a discrete time period becomes the pulse area.
After height and area calculations occur, they are sent to the signal processors.
Compensation, Gating, and Scaling

The digital signal processors (DSPs), located on the acquisition boards and on the master board, perform several important functions including: correcting for spillover between fluorochromes through a series of mathematical calculations (referred to as compensation) window gating scaling and clipping
Scaling
The DSPs take the values determined by the FPGAs and convert them from integer numbers to IEEE-754, 32-bit floating-point numbers. When calculating area, the electronics add all data points under the pulse, in effect increasing the resolution from 16,384 maximum levels of measurement (14 bits) to close to 16,000,000. For a typical pulse of 2-3 microseconds in width, this is equivalent to approximately 18 bits (262,144 levels). In order to normalize the height to the area measurement, the electronics multiples the height by 16 (16,384 x 16 = 262,144). To more finely adjust the area to match the height scale, the DSP applies a floating point scale factor to the area measurement. After the scale factor is applied, the DSP checks the number and limits it (clips) to 262,143. 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.
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Embedded Computer
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 Option White Paper.
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126
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
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Cytometer Supplies
Installation Kit
The cytometer ships with the following items, many of which are used during installation. Use these part numbers to order replacements.
Item Fluid filter 1/4-in. elbow couplings (2) 6-ft cordset for US power (15A, 515P/320-C13) 2.5-m cordset for Australian power (10A C13) 2.5-m cordset for European power (10A C13) 2.5-m cordset for UK power (10A C13 R/A) Filter bypass assembly (3) Bal seal for SIT (6) Floppy disk with saved cytometer.dat file 12 x 75-mm tubes (bag of 125) 10-L waste tank Vented cap for waste tank (pack of 12) Waste cap baffle Spare fluidics cart fuses (2): 6.3-A 250V Slo-blo Type T Loader cover (Loader option only) Part No. 331394 333072 337219 335696 335697 335698 33576007 343509 none 343675 340261 338922 338505 90069-24 338505
128
Other Replacement Parts

Item Sheath sensor probe (5-level) Waste sensor probe (6-level) Auxiliary sensor probe (1-level) Air filter (side door) Bypass tubing Bal seal retainer Carousel kit, carousels 14 Carousel kit, carousels 58 Carousel kit, carousels 912 Carousel kit, carousels 1316 Part No. 334914 334915 334911 336303 336768 336999 332727 332728 332729 332730
Bar Code Reader Parts

Item 2D bar code reader Stand (bar code reader) Part No. 344025 344026
Appendix B: Supplies and Replacement Parts
129
Consumables
Cytometer Setup
Particle BD FACS 7-color setup beads Supplier BD Biosciences (877) 232-8995 Catalog No. 335775
Reagents
Reagent BD FACSFlow sheath fluid Supplier BD Biosciences (877) 232-8995 Catalog No. 340398 (US and Latin America) 342003 (other countries) BD FACSClean solution BD FACS shutdown solution Monoclonal antibodies BD FACS lysing solutionb BD Biosciences BD Biosciences BD Biosciences BD Biosciences 340345 334224
a
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
130
Labware
Item 5-mL polystyrene test tubes, 12 x 75-mm (BD Falcon tubes) uncapped, 125 per bag capped, 125 per bag capped, 25 per bag with cell-strainer cap, 25 per bag Supplier BD Biosciences (877) 232-8995 352052 352054 352058 352235 Catalog No.
Appendix B: Supplies and Replacement Parts
131
132
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.
133
Cytometer Specifications
Dimensions Height: 63.5 cm (25 in.) Width: 90.2 cm (35.5 in.) Depth: 61 cm (24 in.) Workspace dimensions Height (with flow cell access door open): 85 cm (33.5 in.) Unit designed to fit lab bench 55.9 cm (22 in.) depth. Operational clearances, cytometer Left side: 30 cm (11.8 in.) between unit and other objects or wall to permit proper air flow and access to the main power button and circuit breaker Right side: 30 cm (11.8 in.) between unit and other objects or wall to permit proper air flow Top: 22.5 cm (8.9 in.) between unit and other objects or wall to permit opening of flow cell access door Weight 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 (5060 Hz) Current: 5A at 115 VAC 2.5A at 230 VAC Power consumption 500 W
134
Environment
Storage temperature Operating temperature Operating relative humidity Noise level Facilities 140C (34104F) 1530C (5986F) 580% (noncondensing) 62 dBA No special room requirements
Performance
Fluorescence threshold sensitivities Forward and side scatter sensitivity Forward scatter sensitivity Side scatter sensitivity FITC <100 MESF PE <50 MESF Sensitivity enables the resolution of platelets from noise 1 micron 0.5 micron
Optics
Laser Specifications
The following Class 3B lasers are mounted on the BD FACSCanto cytometer.
Wavelength (nm) 488 633 Power (mW) 20 17
Manufacturer Coherent JDS Uniphase
Model Sapphire 488-20 1144-P
These lasers are contained within the cytometer, therefore the BD FACSCanto flow cytometer is a Class I (1) laser product.
Appendix C: Technical Specifications
135
Excitation Optics
Optical platform Beam geometry Fixed optical assembly Blue and red laser: 9 m x 65 m elliptical beam
Emission Optics
Collection lens Optical gelcoupled to flow cell Numerical aperture (NA) = 1.2 Fluorescence detection 6 photomultiplier tube detectors: Four wavelength ranges detected from 488-nm laser: 750810 nm (PE-Cy7) >670 nm (PerCP-Cy5.5) 564606 nm (PE) 515545 nm (FITC) Two wavelength ranges detected from 633-nm laser: 750810 nm (APC-Cy7) 650670 nm (APC) Forward scatter detection Side scatter detection Photodiode with 488/10 bandpass filter PMT with 488/10 bandpass filter
136
Fluidics
General operation Sheath consumption Integrated fluidics cart with automated startup, shutdown and cleaning cycles 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 L/min Medium = 60 L/min High = 120 L/min Sample acquisition rate Recommended maximum particle size 10,000 events/sec with <10% abort rate 50 m
Signal Processing
Workstation resolution Data acquisition channels Fluorescence compensation Pulse processing Time Channel threshold 262,144-channel resolution 8 parameters: 6 fluorescent and 2 scatter parameters No limit to inter- and intra-beam compensation Height, area, and width measurements available for any parameter (BD FACSDiva software) Can be correlated to any parameter Available for any parameter from all lasers
137
Fluidics Cart Specifications

Dimensions Height: 66 cm (26 in.) Width: 81.3 cm (32 in.) Depth: 66 cm (26 in.) Operational clearances Fluidics cart, side air vent: 20 cm (7.9 in.) between air vent and other objects or wall to permit proper air flow Fluidics cart, door air vent: 20 cm(7.9 in.) between door and other objects or wall to permit proper air flow Weight 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 BD FACSClean solution cubitainer BD FACS shutdown solution cubitainer Waste tank 20 L 5L 5L 10 L
138
Loader Specifications
Carousel compatibility Tube compatibility Carousel 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 Tube sample volume 1.07 mL Loader carousels, numbers 116
139
140
Index
A
adapter lever 17 air filter, replacing 60 lock, removing 72 alcohol, isopropyl 75 alignment guide pin 34 arrays, detector 21 aspirator arm 17 assistance, technical xi automatic mode 36, 41 auxiliary air supply switch cap, waste tank 55 carousel rack about 35 installing 38 removing 37 troubleshooting 103 tubes, compatible 35, 139 changing cubitainers 68 circuit breaker cytometer 24, 84 fluidics cart 27 resetting 98 Clean Flow Cell 66, 77 cleaning barcode reader 48 external surfaces 43, 75 flow cell 66, 77 fluidics for storage 80 fluids 31, 75 Loader 43 SIT 77 compensation theory 118120 components FACSCanto system 14 fluidics cart 25 Loader 34 connecting power cords 24 cover, Loader 34, 38, 128 cubitainer about 26 changing 68
27
B
Bal seal 80 bandpass (BP) filters 114 barcode reader about 30 cleaning 48 installing 46 barcode symbologies 49 bleeding filters 57 breaker, main circuit 24 Bubble Filter Purge 66, 78 button, power 24 bypass tubing, installing 72, 73
C
cables, reconnecting 85
141
cytometer circuit breaker 84 cleaning 75 components 15 Loader mode 36, 41 manual mode 39 not responding to software power 24 QC particles 130 specifications 134, 138 supplies 128
98
D
decontamination 59 De-gas Flow Cell 76 detector arrays 21 diode, forward scatter 18 doors access 15 side door 60 dry, sample runs 102
E
electronics 24, 120 ethanol 75
F
fiber optics 20
filters air, replacing 60 bandpass 114 bubble 78 changing 63 fluidic 25 longpass 113 optical theory 113 purging 57 removing an air lock 72 waste tank cap 55 flow cell about 17, 18, 20 access door 15 cleaning 66, 77 problems 96, 97 removing bubbles 76 fluidics about 106 level sensors, replacing 88 long clean 59 priming 71 system components 16 fluidics cart components 25 controls 27 dimensions 138 fuses, replacing 91 maintenance 54, 68 reconnecting tubing 85 troubleshooting 98, 101 fluids leaks cytometer 97 fluidics cart 101 SIT 100 not recommended 75 required 31 fluorescence 112
142
fluorochromes emission spectra 112 for 488-nm and 633-nm lasers spectral overlap and 118 focusing lens 20 forward scatter (FSC) about 111 diode 18 fuses, replacing 91
19
lifter, tube 34 Loader about 28 adapting cytometer 36, 41 cleaning 43 components 34 long clean 59 longpass (LP) filters 113
M G
guide, tube 34 maintenance barcode reader 48 Loader 43 scheduled 52 unscheduled 66 manual mode 39 modes automatic loading 36, 41 manual loading 39
I
installation kit 128 installing air filter 60 barcode reader 46 bypass tubing 73 carousel rack 38 cubitainers 68 fluidics level sensors 88 fuses 91 interlock, laser path 20 isopropyl alcohol 75
O
obscuration bar 18, 22 octagon, optics 21, 23 optical sensors 34 Opticon LG2 Imager 46 optics access door 15 components 18 fiber 20 filters, default 23 filters, theory 113 optical pathway 20 ordering supplies 127
L
label thickness 139 lasers about 18 interlock 20 power 24 specifications 135 troubleshooting 99 leaks cytometer 97 fluidics cart 101 SIT 100 level sensor 88
P
photodiode 18, 22 photomultiplier tubes (PMTs) pin, alignment 34 21
Index
143
power button 15, 24 cytometer 24 laser 24 priming, fluid lines prisms 20 purging filters 57
supplies, cytometer 128 symbologies, barcode 49
71
T
technical assistance xi technical specifications 139 trigon, optics 21, 23 troubleshooting 102 tube guide 34 tubes compatible 35 lifter 34 sample volume 139
R
rack, carousel 139 removing carousel rack 37 replacing air filter 60 fuses 91
U
unscheduled maintenance 66
S
sample injection tube (SIT) about 16 cleaning 77 problems 97, 99, 100 scatter, light 111 scheduled maintenance 52 seal, Bal 80 sensors, fluidics 88 side door, cytometer 15 side scatter (SSC) 111 specifications 139 cytometer 134 fluidics cart 138 spindle 35
V
voltage, fluidics cart fuses 93
W
waste container 26 cap 55 emptying 54
144

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