FACSCanto II Instructions for Use Rev A June 2007

© 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 manual 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 manual 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. Adaptive Server Anywhere is a trademark of Sybase, Inc. 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. 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. 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

640773 Rev. A

5/06

Initial release

642098 Rev. A

9/06

Incorporate changes for filing

642239 Rev. A

6/07

Updated for BD FACSDiva software v6.0

Contents About These Instructions Conventions... Chapter 1: Introduction

xi xii 13

System Components and Theory of Operation...

14

Flow Cytometer Components...

15

Fluidics Cart...

22

BD FACS Loader (Optional)...

25

Barcode Reader (Optional)...

27

System Requirements...

28

Chapter 2: Software Windows and Toolbars

31

BD FACSCanto Clinical Software Workspace...

32

BD FACSCanto Toolbars...

33

BD FACSDiva Software Workspace...

34

Chapter 3: Barcode Reader Option

39

Installing and Using the Barcode Reader...

40

Cleaning the Barcode Reader...

42

Barcode Symbologies...

43

1D Barcode Symbologies...

43

2D Barcode Symbologies...

44

v

Chapter 4: Starting Up

45

Chapter 5: Cytometer QC and Setup

49

Performing Automated Setup...

50

Running Setup Using Manual Loading...

51

Running Setup Using the Loader...

61

Optimizing with BD FACSCanto Clinical Software...

64

Maintaining User-Specific Optimization Settings...

70

Using Cytometer Controls...

71

Reviewing Levey-Jennings Reports...

74

Optimizing with BD FACSDiva Software...

76

Verifying Cytometer Configuration and User Preferences...

77

Creating the Experiment...

78

Applying the Setup Results...

81

Creating Compensation Controls...

82

Optimizing Cytometer Settings...

83

Calculating Compensation...

87

Chapter 6: Running Samples with BD FACSCanto Clinical Software

vi

89

Running an Acquisition Worklist...

90

Entering Information into a Worklist...

91

Running a Process Control...

92

Acquiring Samples...

93

Options During Acquisition...

99

Importing a Worklist from SPA Software...

107

Reviewing an Analysis Worklist...

108

Creating a New Analysis Worklist...

108

Reviewing a Worklist...

111

Logging Out...

113

BD FACSCanto II Instructions for Use

Chapter 7: Running Samples with BD FACSDiva Software

115

Setting Up the Global Worksheet...

116

Recording Data...

118

Importing a Worklist from SPA Software...

120

Analyzing Data...

122

Reusing the Analysis...

125

Saving the Analysis...

125

Logging Out...

127

Chapter 8: Using the Loader with BD FACSDiva Software

129

Getting Ready...

130

Assigning Carousels and Verifying Run Settings...

132

Preparing the Loader...

136

Running Samples...

139

Skipping or Re-Running Samples...

141

Adding Tubes to an Existing Carousel...

144

Re-running a Carousel...

144

Stopping a Run...

144

Running a Single Tube...

145

Running Cleaning Tubes on the Loader...

146

Chapter 9: Shutting Down

147

BD FACSCanto Clinical Software...

147

BD FACSDiva Software...

149

Chapter 10: Maintenance

153

Scheduled Maintenance...

154

Emptying the Waste Container...

156

Purging the Fluidics Filters...

158

Decontaminating the Fluidics System (Long Clean)...

160

Replacing the Air Filter...

161

Replacing Fluidics Filters...

162

Contents

vii

Unscheduled Maintenance...

164

Changing a Cubitainer...

166

Cleaning External Surfaces...

171

Removing Bubbles from the Flow Cell...

172

Cleaning the Flow Cell...

173

Purging the Bubble Filter...

174

Decontaminating the Fluidics System for Storage...

175

Replacing the Bal Seal...

175

Reconnecting the Fluidics Cart Tubing...

181

Replacing the Fluidics Level Sensors...

183

Replacing the Fluidics Cart Fuses...

186

Chapter 11: Troubleshooting

viii

189

Cytometer Troubleshooting...

190

Loader Troubleshooting...

195

Fluidics Cart Troubleshooting...

198

BD FACSCanto Clinical Software Troubleshooting...

199

BD FACSCanto Software General Issues...

199

BD FACSCanto Software Setup Wizard Messages...

203

BD FACSCanto Software Setup Report Failure Messages...

205

BD FACSCanto Software Levey-Jennings Errors and Messages...

207

BD FACSCanto Software Acquisition...

207

BD FACSCanto Software TBNK Analysis QC Messages...

210

BD FACSCanto Software Four- and Six-Color TBNK...

212

BD FACSDiva Software Troubleshooting...

215

BD FACSDiva Software General Issues...

215

BD FACSDiva Software Cytometer Setup...

219

BD FACSDiva Software Acquisition

...

220

BD FACSDiva Software Analysis...

224

Display Troubleshooting...

225

BD FACSCanto II Instructions for Use

Appendix A: Supplies and Replacement Parts

227

Cytometer Supplies...

228

Installation Kit...

228

Other Replacement Parts...

229

Barcode Reader Parts...

229

Consumables...

230

Cytometer Setup...

230

Reagents...

230

Labware...

231

Appendix B: Technical Specifications

233

Cytometer Specifications...

234

Environment...

235

Performance...

235

Optics...

235

Fluidics...

237

Signal Processing...

237

Fluidics Cart Specifications...

238

Capacity...

238

BD FACS Loader Specifications...

239

Index

241

Contents

ix

x

BD FACSCanto II Instructions for Use

About These Instructions These instructions for use contain the information necessary to operate your BD FACSCanto™ II flow cytometer. 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. Use BD FACSCanto clinical software for performing cytometer quality control. 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 II Instructions for Use assumes you have a working knowledge of basic Microsoft® Windows® operation.

xi

Conventions The following tables list conventions used throughout this guide. 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 Risk of electric shock Laser radiation Biological risk

a. Although these symbols appear in color on the cytometer, they are in black and white throughout this document; their meaning remains unchanged.

Table 2 Text and keyboard conventions

xii

Convention

Use

! Tip

Highlights features or hints that can save time and prevent difficulties

Italics

Italics are used to highlight book titles and new or unfamiliar terms on their first appearance in the text.

>

The arrow indicates a menu choice. For example, “choose File > Print” means to choose Print from the File menu.

Ctrl-X

When used with key names, a dash means to press two keys simultaneously. For example, Ctrl-P means to hold down the Control key while pressing the letter p.

BD FACSCanto II Instructions for Use

1 Introduction 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.

13

System Components and Theory of Operation The BD FACSCanto II system consists of a flow cytometer, a self-contained fluidics cart, and the BD FACSCanto II workstation. System options include an automated sample loader and a barcode reader.

14

BD FACSCanto II Instructions for Use

Flow Cytometer Components 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

Do not place heavy objects on top of the cytometer at any time; doing so could cause alteration of data.

Chapter 1: Introduction

15

Fluidics Components

flow cell

tubing

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

Bal seal access

sample injection tube (SIT)

aspirator arm aspirator arm bar

Fluidics Theory of Operation When you install tubes onto the sample injection tube (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.

16

BD FACSCanto II Instructions for Use

Optics Components and Theory of Operation 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.

where lasers intercept sample stream

obscuration bar

flow cell

FSC diode

From the flow cell, laser-excited and scattered light is routed to the detector arrays, which consist of photomultiplier tubes (PMTs) arranged in one octagon and one trigon (a 4-2 optical configuration).

Chapter 1: Introduction

17

In the 4-2 configuration, the octagon contains five PMTs and detects light excited and scattered by the 488-nm (blue) laser. One PMT in the octagon collects side scatter (SSC) signals. The trigon contains two PMTs and detects light excited by the 633-nm (red) laser. The 4-2 configuration octagon and trigon arrays have the filter and mirror combinations shown in Figure 1-2, and Table 1-1 on page 19. Figure 1-2 Trigon and octagon detector arrays (4-2 configuration) trigon

blue-laser signal PMT longpass mirror

bandpass filter

red-laser signal

18

BD FACSCanto II Instructions for Use

octagon

Table 1-1 Trigon and octagon optical filters (4-2 configuration) Detector Array (Laser)

PMT Position

LP Mirror

BP Filter or LP Mirror

Intended Dye

Octagon (488-nm blue laser)

A

735

780/60

PE-Cy™7

B

655

670

PerCP-Cy™5.5 or PerCP

C

610

blank optical holder

-

D

556

585/42

PE

E

502

530/30

FITC

F

blank optical holder

488/10 and pinhole

SSC

G

blank optical holder

blank optical holder

-

H

-

blank optical holder

-

A

735

780/60

APC-Cy7

B

685

blank optical holder

-

C

-

660/20

APC

Trigon (633-nm red laser)

Blank optical holders do not contain optical filters. They are used in the trigon and octagon to prevent unwanted light from interfering with fluorescence signal.

Chapter 1: Introduction

19

Electronics Components 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. NOTICE If a power failure occurs during a run, sample can leak from the SIT. To prevent a biohazardous spill, place an empty tube on the SIT. Once power is restored, remove the tube and perform a fluidics startup before resuming the run. Figure 1-3 Flow cytometer power panel system power button power out to fluidics cart communication cable to fluidics cart 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. Acquisition indicator lights are located on the flow cell access cover on the front of the cytometer (see Figure 1-4 on page 21). Each light corresponds to a detector in the collection optics subsystem, and blinks when the signal at that detector reaches a preset level. Acquisition threshold levels (set using the software) override the presets. See Figure 1-5 on page 21. Lights are activated only when the system is acquiring data, and only the indicators corresponding to currently active parameters will blink.

20

BD FACSCanto II Instructions for Use

CP -C y5 .5 PE -C or y7 Pe rC P PE -Te xa sR AP ed C (5 -3 on AP ly) CCy 7 Al ex aF luo Am r7 00 Cy an (5 -3 ( 4Pa on 2cif ly ) 2 ic o nl Bl y) ue (4 -2 -2 on ly)

Pe r

PE

C FIT

C SS

FS

C

Figure 1-4 Detector order of acquisition indicator lights

The acquisition indicator lights can be switched off. The on/off switch is located inside the flow cell access cover. Figure 1-5 Acquisition indicator light switch flow cell access cover

acquisition indicator light switch

Chapter 1: Introduction

21