BD FACSCanto Reference guide

Type
Reference guide
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Part No. 642241 Rev. A
June 2007
For In Vitro Diagnostic Use
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BD FACSCanto
Flow Cytometer
Reference Manual
© 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
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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 user’s 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 numérique de la classe A respecte toutes les exigences du Réglement sur le matériel 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/user’s responsibility to install and maintain up-to-date virus protection software.
BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation.
BD Biosciences is not liable for any claims related to or resulting from buyer/user's failure to install and maintain
virus protection.
History
Revision Date Change Made
336928 Rev. A 1/04 Initial release
337969 Rev. A 4/04 Updated for CE IVD release
338619 Rev. A 9/04 Updated for BD FACSDiva software v4.1
343370 Rev. A 9/05 Updated for BD FACSCanto clinical software v2.0 and the Opticon LG2 Imager barcode reader
640801 Rev. A 5/06 Updated for BD FACSDiva software v5.0 and BD FACSCanto clinical software v2.1
642241 Rev. A 6/07 Updated for BD FACSDiva software v6.0
v
Contents
About This Manual ix
Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Chapter 1: Introduction 13
Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Fluidics Cart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Computer Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
BD FACS Loader (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Barcode Reader (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Chapter 2: BD FACS Loader Option 33
Loader Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Using the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Changing Operational Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Maintaining the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Cleaning External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
vi BD FACSCanto Flow Cytometer Reference Manual
Chapter 3: Barcode Reader Option 45
Installing and Using the Barcode Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Cleaning the Barcode Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
1D Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2D Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Chapter 4: Maintenance 51
Scheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Emptying the Waste Container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Purging the Fluidics Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Decontaminating the Fluidics System (Long Clean) . . . . . . . . . . . . . . . . 59
Replacing the Air Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Replacing Fluidics Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Unscheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Changing a Cubitainer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Removing an Air Lock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Cleaning External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Removing Bubbles from the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Cleaning the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Purging the Bubble Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Decontaminating the Fluidics System for Storage . . . . . . . . . . . . . . . . . . 80
Replacing the Bal Seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Resetting the Cytometer Circuit Breaker . . . . . . . . . . . . . . . . . . . . . . . . . 84
Reconnecting the Ethernet and RS232 Cables . . . . . . . . . . . . . . . . . . . . . 85
Reconnecting the Fluidics Cart Tubing . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Replacing the Fluidics Level Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Replacing the Fluidics Cart Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Contents vii
Chapter 5: Troubleshooting 95
Cytometer Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Fluidics Cart Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Loader Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Appendix A: Technical Overview 105
Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Fluidics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Optics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Spillover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Electronics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Pulse Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Appendix B: Supplies and Replacement Parts 127
Cytometer Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Installation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Other Replacement Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Bar Code Reader Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Cytometer Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
viii BD FACSCanto Flow Cytometer Reference Manual
Appendix C: Technical Specifications 133
Cytometer Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Signal Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Fluidics Cart Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Loader Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Index 141
ix
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 non–application 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.
x BD FACSCanto Flow Cytometer Reference Manual
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 symbols
a
a. Although these symbols appear in color on the cytometer, they are in black and white throughout this
reference manual; their meaning remains unchanged.
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
Table 2 Text and keyboard conventions
Convention Use
; Tip
Features or hints that can save time or prevent difficulties
NOTICE
Provides information that supplements the topic material
Italics 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.
About This Manual xi
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.
xii BD FACSCanto Flow Cytometer Reference Manual
13
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 facilities—it plugs into a standard wall
outlet, uses air-cooled lasers, and provides its own air pressure and vacuum for
the fluidics and waste. You can prepare samples on the BD FACS™ Sample Prep
Assistant II and import the sample prep worklist.
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
14 BD FACSCanto Flow Cytometer Reference Manual
Intended Use
The BD FACSCanto system is intended for use as an In Vitro Diagnostic device
for identification and enumeration of lymphocyte subsets in human cells in
suspension for flow cytometry.
System Components
The BD FACSCanto system consists of a benchtop flow cytometer, a self-
contained fluidics cart, and the BD FACSCanto workstation.
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
Chapter 1: Introduction 15
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).
The BD FACSCanto flow cytometer consists of a fluidics subsystem, an optics
subsystem, and an electronics subsystem. For a more in-depth discussion of
sample
injection
tube
data ports
side door
fluidics cart
connections
flow cell
access door
optics
access door
power button
16 BD FACSCanto Flow Cytometer Reference Manual
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.
sample
injection
tube (SIT)
aspirator arm
tubing
tube guide
(Loader option only)
aspirator arm bar
flow cell
adapter lever
Chapter 1: Introduction 17
The following table briefly describes these components.
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.
flow cell where laser beam intercepts particles
tubing brings sheath and cleaning fluids to and aspirates waste from flow cell
SIT hollow metal tube that brings sample to flow cell
adapter lever lever used to change SIT from manual to automatic loading
aspirator arm movable waste aspiration port
aspirator arm
bar
metal bar used to push aspirator arm away from SIT during manual
loading
tube guide helps guide tube onto the SIT (Loader operation only)
18 BD FACSCanto Flow Cytometer Reference Manual
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
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.
FSC diode
w
h
ere
l
asers intercept
sample stream
flow cell
obscuration bar
Chapter 1: Introduction 19
The BD FACSCanto cytometer uses low-powered air-cooled and solid state lasers
that do not have special power and cooling requirements.
Laser
Wavelength
(nm)
Commonly Used Fluorochromes
Coherent® Sapphire™
Solid State
488 (blue) FITC, PE, PerCP,
PerCP-Cy™5.5, PE-Cy™7
JDS Uniphase™ HeNe
Air Cooled
633 (red) APC, APC-Cy7
20 BD FACSCanto Flow Cytometer Reference Manual
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
When the flow cell access door opens, an interlock shutters the laser light and
blocks its pathway for safety reasons.
1
2
3
4
5
6
interlock flow cell
prisms red He-Ne laser
focusing lens blue solid-state laser
1
4
2 5
3 6
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BD FACSCanto Reference guide

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Reference guide

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