FACSCanto II Reference Manual Rev A May 2009

© 2006, 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, the BD logo, BD Calibrite, BD FACS, BD FACSCalibur, BD FACSCanto, BD FACSDiva, BD FACSFlow, BD Falcon, BD Multiset, BD Multitest, and BD Trucount are trademarks of Becton, Dickinson and Company. JDS Uniphase is a trademark of JDS Uniphase, Inc. Alexa Fluor and Texas Red are registered trademarks, and Pacific Blue is a trademark of Molecular Probes, Inc. iFLEX is a trademark of Point Source, Ltd. 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 PE and APC: US 4,520,110; 4,859,582; 5,055,556; Europe 76,695; Canada 1,179,942 PerCP: US 4,876,190 Cy5.5 and Cy7: US 5,268,486; 5,486,616; 5,569,587; 5,569,766; and 5,627,027 Pe-Cy7: US 4,542,104 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 the matériel brouilleur du Canada.

Notice BD Biosciences delivers software and workstations that are intended for running the instruments 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

640806 Rev. A

5/06

Initial release

Contents About This Manual

ix

Conventions...

x

Technical Assistance...

xi

Chapter 1: Introduction

13

Intended Use...

14

System Components...

15

Flow Cytometer...

16

Fluidics Cart...

26

Computer Workstation...

29

BD FACS Loader (Optional)...

29

Barcode Reader (Optional)...

30

System Requirements...

31

Chapter 2: BD FACS Loader Option

33

Loader Components...

34

Using the Loader...

36

Opening and Closing the Loader Doors...

36

Acquiring with the Loader...

37

Changing Operational Modes...

40

Maintaining the Loader...

42

Spill Containment Area...

42

Cleaning External Surfaces...

43

v

Chapter 3: Barcode Reader Option 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

vi

45

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...

74

Removing Bubbles from the Flow Cell...

76

Cleaning the Flow Cell...

76

Purging the Bubble Filter...

78

Decontaminating the Fluidics System for Storage...

79

Replacing the Bal Seal...

79

Resetting the Cytometer Circuit Breaker...

85

Reconnecting the Ethernet and RS232 Cables...

86

Reconnecting the Fluidics Cart Tubing...

86

Replacing the Fluidics Level Sensors...

89

Replacing the Fluidics Cart Fuses...

92

BD FACSCanto II Flow Cytometer Reference Manual

Chapter 5: Troubleshooting

95

Instrument 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...

110

Fluorescence...

111

Optical Filters...

112

Detectors...

114

Detector Arrays...

114

Spillover...

117

Electronics System...

119

Pulses...

120

Pulse Measurements...

121

Appendix B: Optics Configurations

125

4-2 Optics Configuration...

126

4-2-2 Optics Configuration...

130

5-3 Optics Configuration...

134

Contents

vii

Appendix C: Supplies and Replacement Parts Instrument Supplies...

140

Installation Kit...

140

Other Replacement Parts...

141

Bar Code Reader Parts...

141

Consumables...

142

Instrument Setup...

142

Reagents...

142

Labware...

143

Appendix D: Technical Specifications

145

Cytometer Specifications...

146

Environment...

147

Performance...

147

Optics...

147

Fluidics...

149

Signal Processing...

149

Fluidics Cart Specifications...

150

Capacity...

150

BD FACS Loader Specifications...

151

Index

viii

139

153

BD FACSCanto II Flow Cytometer Reference Manual

About This Manual This manual contains reference information about the BD FACSCanto™ II flow cytometer and the BD FACS™ Loader. Operating instructions are contained in the BD FACSCanto II Instructions for Use. Most instrument 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 instrument quality control. Each software package has its own reference manual, provided on the documentation CD. BD Biosciences recommends that first-time users of this instrument take advantage of operator training offered with the sale of every new instrument. The BD FACSCanto II 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 instrument, they are in black and white throughout this reference manual; their meaning remains unchanged.

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.

x

BD FACSCanto II Flow Cytometer Reference Manual

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 instrument 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

BD FACSCanto II Flow Cytometer Reference Manual

1 Introduction The BD FACSCanto II system uses a fixed-optics design and advanced digital electronics to support multicolor analysis of up to eight fluorescent markers and two scatter parameters per assay. The instrument 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 15

•

System Requirements on page 31

13

Intended Use The BD FACSCanto II 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.

14

BD FACSCanto II Flow Cytometer Reference Manual

System Components The BD FACSCanto II system consists of a benchtop flow cytometer, a selfcontained fluidics cart, and the BD FACSCanto II workstation. System options include an automated sample loader and a barcode reader.

For a description of system components, see: •

Flow Cytometer on page 16

•

Fluidics Cart on page 26

•

Computer Workstation on page 29

•

BD FACS Loader (Optional) on page 29

•

Barcode Reader (Optional) on page 30

Chapter 1: Introduction

15

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 instrument). Figure 1-1 BD FACSCanto II flow cytometer

flow cell access door side door data ports optics access door

acquisition indicator lights sample injection tube power button fluidics cart connections

The BD FACSCanto II flow cytometer consists of a fluidics subsystem, an optics subsystem, and an electronics subsystem. For a more in-depth discussion of 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 (plenum), and a network of tubing that provides sheath and cleaning fluids to and removes waste from the flow cell.

16

BD FACSCanto II Flow Cytometer Reference Manual

flow cell

tubing

tube sensor

Bal seal access

tube eject cylinder (used with Loader option only) tube guide (Loader option only)

sample injection tube (SIT)

aspirator arm aspirator arm bar

The following table briefly describes these components. flow cell

where laser beam intercepts particles

tubing

brings sheath and cleaning fluids to and aspirates waste from flow cell

SIT

hollow metal tube that brings sample to flow cell

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)

tube eject cylinder

ejects tube (Loader operation only)

tube sensor

detects tube position on SIT

Chapter 1: Introduction

17

When you install tubes onto the SIT, a pump within the fluidics cart pressurizes the 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. SIT cleaning between tubes is automatic when you use BD FACSCanto clinical software. In BD FACSDiva software, SIT cleaning between tubes is automatic unless you disable it by deselecting the SIT Flush checkbox on the Acquisition Dashboard. Do not leave a tube of distilled water on the SIT between sample tubes, or during or after daily shutdown. Never install a tube onto the SIT that contains more than 3 mL of fluid. This will cause the tube sensor to become wet and fail to properly sense tubes.

Flow Cell Once the sample moves into the flow cell, particles move in single file through 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 behind the flow cell access door. For more information, see Fluidics System on page 106.

18

BD FACSCanto II Flow Cytometer Reference Manual

Figure 1-2 Flow cell

obscuration bar

where lasers intercept sample stream

FSC diode

flow cell

Optics The optics system in the BD FACSCanto II 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.

Optics Configurations Several optics configurations are available for the BD FACSCanto II cytometer. The 4-2 configuration can collect four fluorescence signals from a blue laser source and two fluorescence signals from a red laser source. The 4-2-2 configuration adds a violet laser and can collect two fluorescence signals from the violet laser source. The 5-3 configuration uses only the blue and red lasers, but can collect an additional fluorescence signal from each laser source. The excitation and collection optics components for the 4-2 configuration are described in this section. The additional optics configurations and their components are described in Appendix B, Optics Configurations.

Chapter 1: Introduction

19

Excitation Optics The excitation optics consist of lasers, fiber optic cables, beam-shaping prisms, and an achromatic focusing lens, as shown in Figure 1-3. The BD FACSCanto II instrument uses low-powered air-cooled and solid state lasers that do not have special power and cooling requirements. Table 1-1 4-2 configuration fluorochromes Wavelength (nm)

Laser

Commonly Used Fluorochromes

Coherent® Sapphire™ Solid State

488 (blue)

FITC, PE, PE-Cy7, PerCP or PerCP-Cy5.5

JDS Uniphase™ HeNe Air Cooled

633 (red)

APC, APC-Cy7

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 1-3). See also Figure A-3 on page 109. Figure 1-3 Optical pathway (figure shows violet laser available with 4-2-2 configuration only)

3 1

2

4 1 interlock

3

focusing lens

2 prisms

4

flow cell

When the flow cell access door opens, an interlock shutters the laser light and blocks its pathway for safety reasons.

20

BD FACSCanto II Flow Cytometer Reference Manual

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 4-2 configuration collection optics include two detector arrays, which consist of photomultiplier tubes (PMTs) arranged in one octagon and one trigon as shown in Figure 1-4. 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 1-4 Octagon and trigon detector arrays (4-2 configuration) trigon

blue-laser signal

PMT longpass dichroic mirror

bandpass filter

red-laser signal

octagon

Chapter 1: Introduction

21