High Throughput Samplers User's Guide 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 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 © 2007, 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. This product includes software developed by the Apache Software Foundation (apache.org). BD, BD logo, and all other trademarks are property of Becton, Dickinson and Company. Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated. Diskeeper is a registered trademark of Executive Software International. Microsoft and Windows are registered trademarks of Microsoft Corporation. Sybase, Adaptive Server Anywhere, and SQL Anywhere are trademarks of Sybase, Inc or its subsidiaries. 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. 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, may 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 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

338642

10/04

Initial release

640756

3/06

Updated to include BD FACSDiva software, version 5.0, and compatibility with BD FACSCanto and BD FACSCanto II flow cytometers.

642224

6/07

Updated to include BD FACSDiva software, version 6.0; added configuration and performance tracking module; operational improvements; and increased cytometerspecific improvements.

Contents About This Guide

ix

Conventions...

x

Technical Assistance...

xi

Chapter 1: Introduction

13

HTS Hardware Overview...

14

Components...

16

Cytometer Connections...

19

Cytometer Interface Panel...

19

Sample Coupler...

21

Sample Processing...

23

Chapter 2: BD FACSDiva Software Overview

27

Workspace Components...

28

Plate Window...

32

Setup View Components...

33

Setup View Functions...

43

Analysis View Components...

48

Analysis View Functions...

51

Chapter 3: Running Samples

55

Starting Up...

56

Setting Up for Plate-Based Acquisition...

59

v

Creating Experiments...

64

Creating a Folder and an Experiment...

64

Setting Up the Plate...

67

Setting Up the Worksheet...

74

Exporting a Plate as a Template...

76

Assigning Keywords...

77

Preparing for Acquisition...

78

Optimizing Cytometer and HTS Settings...

78

Performing Compensation...

81

Acquiring Data...

82

Pausing the BD High Throughput Sampler...

83

Stopping the BD High Throughput Sampler...

84

Exporting an Experiment as a Template...

84

Analyzing Data...

86

Displaying Keywords...

86

Performing Data Analysis...

90

Maintaining Data...

93

Returning to Tube-Based Acquisition...

94

Returning to Tube-Based Acquisition on the BD LSR II...

94

Returning to Tube-Based Acquisition on the BD FACSCanto and BD FACSCanto II...

97

Shutting Down...

100

Quitting the Software...

101

Chapter 4: Maintenance

vi

103

Daily Maintenance...

104

Daily Cleaning...

104

Cytometer Inspection and Servicing...

109

Monthly Maintenance...

111

Monthly Cleaning...

111

Surface Inspection and Cleaning

114

...

BD High Throughput Sampler User’s Guide

Periodic Maintenance...

114

Replacing the Sample Injection Tubing...

116

Replacing the HTS Sheath Filter...

119

Cleaning the Air Filter...

121

Replacing the Sample Coupler and Tubing...

123

Replacing a Pump Syringe...

125

Replacing the Probe...

129

Replacing Quick-Connector O-Rings...

130

Replacing SIT Protector and O-Ring (BD LSR II Only)...

130

Placing HTS Unit into Long-Term Storage...

132

Unscheduled Maintenance...

136

Homing the Sample Probe...

136

Priming the HTS...

138

Performing a Motion Test...

138

Verifying the Sample Probe Position...

140

Reinitializing the HTS...

142

Inspecting Thumbscrew Fittings...

142

Inspecting Hexagonal Fittings...

143

Declogging the SIT...

143

Chapter 5: Troubleshooting

145

HTS Troubleshooting...

146

Acquisition Troubleshooting...

147

BD FACSDiva Troubleshooting...

149

Appendix A: Consumables and Replacement Parts

151

Cytometer Supplies...

152

Maintenance Log...

154

Contents

vii

Appendix B: Depot Repair Procedures Depot Repair Overview...

158

Removing the HTS Unit...

159

Cleaning the HTS Unit...

162

Decontaminating the Fluidics Manually...

163

Decontaminating External Surfaces...

167

Unpacking the Replacement Unit...

169

Lifting Heavy Objects...

169

Unpacking the HTS Unit...

170

Installing the HTS Unit...

170

Packing the Unit for Shipping...

175

Placing the HTS into Its Shipping Container...

175

Index

viii

157

177

BD High Throughput Sampler User’s Guide

About This Guide This guide describes how to set up and operate the BD™ High Throughput Sampler (HTS) option with the BD™ LSR II, BD FACSCanto™, and BD FACSCanto™ II flow cytometers. You should know how to operate your flow cytometer before using the HTS option. For important safety information, refer to the safety booklet provided with the HTS option. Cytometer function is controlled by BD FACSDiva™ software. In this guide, you will find a description of BD FACSDiva software features specific to the HTS option. BD LSR II, BD FACSCanto, and BD FACSCanto II flow cytometers modified with the HTS can acquire samples from plates or tubes. Even when acquiring samples using the HTS, consult the appropriate cytometer user’s guide for information about flow cytometer operation, daily shutdown, maintenance, and troubleshooting. The BD High Throughput Sampler User’s Guide 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 throughout this guide. Table 1 lists the symbols that are used in this guide 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 Risk of electric shock Biological risk

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

Table 2 Text and keyboard conventions Convention

Use

NOTICE

Describes important features or instructions.

! Tip

x

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 High Throughput Sampler User’s Guide

Technical Assistance For technical questions or assistance in solving a problem: •

Read the section of the user’s guide 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 and serial number

•

any error messages

•

details of recent system performance

For cytometer support from within the US, call (877) 232-8995. For support from within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor. If you need to send your HTS unit back to BD Biosciences for repair, see Appendix B on page 157 for instructions. Note that depot repair procedures might be different outside of the United States. Contact your local BD Biosciences service representative for information for your region.

About This Guide

xi

xii

BD High Throughput Sampler User’s Guide

1 Introduction The BD High Throughput Sampler (HTS) is a compact, high-speed sample loading device for use with the BD LSR II, BD FACSCanto, and BD FACSCanto II flow cytometers. BD FACSDiva software controls the sample loader, providing automated acquisition of samples from a multiwell plate. Automate analysis using the BD FACSDiva batch analysis feature, for an efficient, high-throughput system. The following topics are covered in this chapter: •

HTS Hardware Overview on page 14

•

Components on page 16

•

Cytometer Connections on page 19

•

Sample Processing on page 23

13

HTS Hardware Overview Easy to use and maintain, and highly reliable, the HTS provides the following basic functionality: •

Acquires samples from 96- and 384-well plates (standard depth)

•

Includes two throughput modes: standard and high throughput

•

Minimizes carryover

•

Provides user-definable mixing and sample introduction protocols

•

Is user-installable (software and sampler unit only; initial installation excluded)

•

Supports immunophenotyping assays

The HTS can process a 96-well plate in approximately 44 minutes in standard mode and approximately 15 minutes in high-throughput mode using the default settings listed in Table 1-1. Table 1-1 Default throughput mode settings Standard Mode

High-Throughput Mode

Sample Flow Rate (µL/sec)

1

1

Sample Volume (µL)

10

2

Mixing Volume (µL)

100

50

Mixing Speed (µL/sec)

180

200

Number of Mixes (cycles)

2

2

Wash Volume (µL)

400

200

Approximate Acquisition Time (min)

44

15

Setting

14

BD High Throughput Sampler User’s Guide

For more information, see Loader Settings on page 38. The HTS unit is installed on the cytometer by a BD Biosciences service engineer. Once installed, the HTS enables quick conversion of the flow cytometer from tube- to plate-based acquisition. Figure 1-1 HTS installed on a BD LSR II flow cytometer BD LSR II flow cytometer

BD HTS Unit

Chapter 1: Introduction

15

Components See Figure 1-2 on this page, Figure 1-3 on page 17, and Figure 1-4 on page 18 to familiarize yourself with the specific HTS hardware components for your cytometer. For a description of HTS cytometer connections, see Cytometer Connections on page 19. Figure 1-2 HTS unit-front view (example of HTS)

1

2 3

1

Plate holder-moves left to right and front to back to position plate so the probe can pick up sample

2

Primary pump and valve-enables mixing and aspiration of sample; delivers sample to flow cell in standard mode

3

Power switch and LED indicator for HTS unit

16

BD High Throughput Sampler User’s Guide

Figure 1-3 HTS unit for BD LSR II and BD FACSCanto-rear view 4

1 5

6

3 2

7 8

1

Probe assembly-moves front to back and up and down to transfer sample between plate holder and injection port/wash station

2

Fluidics tubing: sheath (clear) and waste (orange)

3

Sheath filter-filters incoming sheath fluid to HTS unit

4

Injection port/wash station-provides interface for sample injection and probe washing

5

Plate holder-moves left to right and front to back to position plate so the probe can pick up sample

6

Secondary pump and valve-delivers sample to flow cell in highthroughput mode

7

Absorbent pad-collects potential overflow from the injection port/ wash station or drips from the cytometer sample injection tube (SIT)

8

Sample coupler-connector between HTS unit to cytometer SIT

Chapter 1: Introduction

17

Figure 1-4 HTS unit for BD FACSCanto II-rear view

4

1 5 6

7 2 8 3

18

1

Probe assembly-moves front to back and up and down to transfer sample between plate holder and injection port/wash station

2

Fluidics tubing: sheath (clear, with in-line filter) and waste (orange)

3

Door sensor cable connector-cable detects if safety door is open

4

Injection port/wash station-provides interface for sample injection and probe washing

5

Sample coupler-connector between HTS unit to cytometer SIT

6

Plate holder-moves left to right and front to back to position plate so the probe can pick up sample

7

Secondary pump and valve-delivers sample to flow cell in highthroughput mode

8

Overflow reservoir-collects potential overflow from the injection port/ wash station or drips from the cytometer SIT

BD High Throughput Sampler User’s Guide

Cytometer Connections Cytometer Interface Panel Sheath and waste travel between the cytometer and the HTS unit via connectors in the cytometer interface panel (see Figure 1-5, Figure 1-6, and Figure 1-7). The BD LSR II interface panel also includes an acquisition mode switch, which controls pressure at the cytometer sample injection tube (SIT). •

In Tube mode ( ), the droplet containment module (DCM) vacuum functions normally-the vacuum is on when the arm is positioned to the side and off when the arm is centered. Backdripping from the sample injection tube is contained when the DCM sleeve is installed.

•

In Plate mode ( ), the DCM vacuum on the cytometer does not function. Drip containment is provided by the absorbent pad in back of the HTS unit (Figure 1-3 on page 17). [BD LSR II] To keep your cytometer free of drips from potentially biohazardous samples, always switch the cytometer to Tube mode and install the DCM sleeve when you are not acquiring samples using the HTS option. Note that if backdripping does occur, drips are contained by the absorbent pad in back of the HTS unit.

Chapter 1: Introduction

19

Figure 1-5 Cytometer interface panel [BD LSR II] Tube/Plate acquisition mode switch sheath connector waste connector waste tubing

sheath tubing

cytometer support bracket

power cable communication cable

base plate

HTS positioning screw

Figure 1-6 Cytometer interface panel [BD FACSCanto]

communication cable waste tubing

sheath tubing

power cable base plate

20

BD High Throughput Sampler User’s Guide

Figure 1-7 Cytometer interface panel [BD FACSCanto II]

interface/ communication connector waste connector

sheath connector NOTE: The white air connector is used for the pneumatic tube loader. door sensor cable

enclosure

Sample Coupler A sample coupler connects the injection port tubing on the HTS unit to the cytometer sample injection tube (SIT) (Figure 1-8 on page 22). For the BD LSR II, the droplet containment module (DCM) sleeve is replaced by the SIT protector. The SIT protector is a modified sleeve that prevents the sample injection tube from bending during installation of the HTS sample coupler. For the BD FACSCanto and BD FACSCanto II, the sample coupler is installed on the SIT, while the aspirator arm rests against the sample coupler. Flip the aspirator arm bar to the back (as shown in Figure 1-8 on page 22) to ensure it does not come in contact with the HTS probe. This position also ensures that the aspirator arm is able to detect an installed sample coupler.

Chapter 1: Introduction

21