Bio-Rad Bio-Plex Software Upgrades and Conversions User Manual
Bio-Plex Manager™ Software 6.1
User Guide
Bio-Plex Manager™
Software 6.1 User Guide
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive,
Hercules, CA 94547 · 1-800-424-6723
10022815 Rev B
Bio-Plex Manager Software 6.1 User Guide
BIO-RAD TECHNICAL SUPPORT DEPARTMENT
The Bio-Rad Technical Support Department in the U.S. is open Monday through Friday,
5:00 a.m. to 5:00 p.m., Pacific Standard Time. Worldwide technical support is available on
the Web at www.consult.bio-rad.com.
Phone: 1-800-424-6723, option 2
Fax: 1-510-741-5802
Email: LSG.TechServ.US@Bio-Rad.com (U.S.)
LSG.TechServ.Intl@Bio-Rad.com (International)
Web: www.consult.bio-rad.com
NOTICE
No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopy, recording, or any information storage or
retrieval system, without permission in writing from Bio-Rad.
Bio-Rad reserves the right to modify its products and services at any time. This user guide
is subject to change without notice. Although prepared to ensure accuracy, Bio-Rad
assumes no liability for errors or omissions, or for any damage resulting from the
application or use of this information.
The following are trademarks of Bio-Rad Laboratories: Bio-Rad, Bio-Plex, and Bio-Plex
Manager. Luminex, xMAP, and xPONENT are trademarks of Luminex Corporation.
Windows is a trademark of Microsoft Corporation. Pentium is a trademark of Intel
Corporation.
No rights or licenses under any of Luminex Corporation’s patents are granted by or shall
be implied from the sale or acquisition of this Bio-Plex system containing Luminex
technology (the “System”) to you, the end-user. By using this System, you agree that (i) the
System is sold only for use with fluorescently labeled microsphere beads authorized by
Luminex (“Beads”), and (ii) you obtain rights under Luminex’s patents to use this System
by registering this System with Bio-Rad in accordance with the instructions accompanying
this System and by Bio-Plex Manager Software 6.1 User Guidepurchasing a kit containing
Beads.
Copyright © 2001–2011 by Bio-Rad Laboratories. All rights reserved.
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Bio-Plex Manager Software 6.1 User Guide
Table of Contents
Chapter 1. Bio-Plex Suspension Array System Overview . . . . . 1
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
For More Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bio-Rad Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Chapter 2. Bio-Plex Manager™ Software Overview . . . . . . . . . 5
Software Editions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Software Licenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Compatibility with Luminex Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
General Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Types of Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Key Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Chapter 3. Software Installation . . . . . . . . . . . . . . . . . . . . . . . . 11
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Required Screen Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Installing the Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Microsoft.NET. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Bioplexdata.mdb File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Luminex LXR Directory and Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Hardware Protection Key (HASP Key). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Uninstalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Chapter 4. Starting the System . . . . . . . . . . . . . . . . . . . . . . . . . 15
Starting Bio-Plex Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Connecting with the Array Reader and Microplate Platform . . . . . . . . 16
Communication between the Array Reader and Microplate Platform . 17
Disconnecting and Reconnecting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
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Bio-Plex Manager Software 6.1 User Guide | Table of Contents
Disconnecting with Sleep Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Menu and Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Quick Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sample Needle Adjustment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Chapter 5. Controlling the System . . . . . . . . . . . . . . . . . . . . . . 25
Bio-Plex MCV Plate IV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Start Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Optics Warm Up and Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Opening the Calibration Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Selecting/Entering Calibration Control Numbers . . . . . . . . . . . . . . . . . 30
Setting Up the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Performing the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Logging the Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Start Up and Calibrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Wash Between Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Remove Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Unclog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Validation Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Validation Kit Control Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Setting Up a Validation Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Control Number Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Validation Type Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Performing a Validation Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Validation Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Validation Log. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Platform Heater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Instrument Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Eject/Retract Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Additional Instrument Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Cancel Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Instrument Operations Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
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Bio-Plex Manager Software 6.1 User Guide
Chapter 6. Preparing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . 53
Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Creating/Opening Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Saving Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Reducing the File Size of Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Multi-Assay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Uses for Multi-Assay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Creating a Multi-Assay Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Changing a Protocol from Single to Multi-Assay . . . . . . . . . . . . . . . . . 57
Protocol Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Step 1. Describe Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Step 2. Select Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Step 3. Format Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Plate Groupings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Step 4. Enter Standards Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Standard Lots. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Step 5. Enter Controls Info (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . 96
Step 6. Enter Sample Info (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Chapter 7. Running Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Run Protocol Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Bead Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Sample Timeout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Reservoir Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Skip Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Standard Curve Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Advanced Run Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Bead Map Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Sample Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Save Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Sampling Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Doublet Discriminator Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Plate Loading Guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Running the Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Status Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Manually Stopping a Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Generating Results from a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Histogram and Bead Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Bead Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
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Bio-Plex Manager Software 6.1 User Guide | Table of Contents
Bead Map Display Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Raw Data Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Bead Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Sampling Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Raw Data Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Set Number Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Other Table Formatting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Copying the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Rerun/Recovery Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Chapter 8. Using the Security Edition . . . . . . . . . . . . . . . . . . . 125
Bio-Plex Manager™ Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Background on 21 CFR Part 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Standard Mode vs. Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Installing and Starting Bio-Plex Manager Security Edition . . . . . . . . . . . . . . . 127
Users, Passwords, and User Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
User Level Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
User Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Enabling and Disabling Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
User Authentication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Electronic Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
File Security and Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Calibration, Validation, and Instrument Operations Logs . . . . . . . . . . . . . . . . 131
Secure Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Unsigned Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . 131
Signed Protocol and Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Document ID Number and Signature . . . . . . . . . . . . . . . . . . . . . . . . . 134
Adjusting the Number of Unknown Samples in a Protocol . . . . . . . . 135
Generating a Results File from a Protocol File. . . . . . . . . . . . . . . . . . 136
Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Viewing the Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Protected Directories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Locking Bio-Plex Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Logging Off. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Chapter 9. Analyzing the Results . . . . . . . . . . . . . . . . . . . . . . 143
Results Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Opening a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Saving a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
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Bio-Plex Manager Software 6.1 User Guide
Reducing the Size of a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Results Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Custom Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Viewing/Changing the Protocol Settings . . . . . . . . . . . . . . . . . . . . . . 148
Changing the Doublet Discriminator Gate Range . . . . . . . . . . . . . . . 149
Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Setting the Plate ID. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Exporting the Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Raw Data Table Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Printing the Raw Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Report Table Column Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Obs/Exp * 100 Column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Concentration in Range Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Showing/Hiding Outliers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Resizing the Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Context Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Sorting Report Table Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Copying the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Printing the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Exporting the Report Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Export Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Export a Subset of the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Export Preferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Use Multiple Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Exclude Standard Curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Exclude Column Headers/Footers . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Exclude Table Error Codes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Regression Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Linear vs. Logistic Regression Methods . . . . . . . . . . . . . . . . . . . . . . 173
Copying the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Exporting the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Printing the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Graphing Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Choosing Graph Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Viewing Information within Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Adding New Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Editing an Existing Graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
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Bio-Plex Manager Software 6.1 User Guide | Table of Contents
Deleting a Graph. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Exporting Graphs to Other Applications . . . . . . . . . . . . . . . . . . . . . . 184
Adjusting Your Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Printing Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Document Export Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Bio-Plex XML Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Output CSV File Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Using Stylesheets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Command Line Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Exporting the Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Importing Luminex xPONENT Output Files. . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Exporting Results Data to Bio-Plex Data Pro™ . . . . . . . . . . . . . . . . . . . . . . . 195
Chapter 10. Standard Curve Optimizer . . . . . . . . . . . . . . . . . . 199
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Standard Curve Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Concentration in Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Determining Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Recommended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Standard Curve Optimizer Report . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Residual Variance and Fit Probability. . . . . . . . . . . . . . . . . . . . . . . . . 204
Logistic Weighting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Chapter 11. Data Normalization . . . . . . . . . . . . . . . . . . . . . . . 207
Normalization Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Selecting Internal Controls/Housekeeping Genes . . . . . . . . . . . . . . . 208
Assigning a Control Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Report Table Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Predefined Normalization Report Schemes . . . . . . . . . . . . . . . . . . . . 211
Normalization Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Calculations Based Upon Your Settings . . . . . . . . . . . . . . . . . . . . . . 212
Normalization Factor Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Security Edition: User Access by Function . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Normalization Formulas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Norm Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
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Bio-Plex Manager Software 6.1 User Guide
Norm Ratio’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Norm Ratio’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Normalized Fluorescence Values (Norm FI) . . . . . . . . . . . . . . . . . . . . 225
Ratio (Simple) and the Grouping Function . . . . . . . . . . . . . . . . . . . . . 226
Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Reporter Fluorochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
Microsphere Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Microsphere Dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Probe Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Bath Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Enumeration of Microsphere Suspensions . . . . . . . . . . . . . . . . . . . . 230
Microsphere Separation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Microsphere Agitation During Assay . . . . . . . . . . . . . . . . . . . . . . . . . 231
Microsphere Stability and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Software Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Reporting Problems to Bio-Rad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Report Table Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
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Bio-Plex Manager Software 6.1 User Guide | Table of Contents
x
Bio-Plex Manager Software 6.1 User Guide
1 Bio-Plex Suspension
Array System Overview
The Bio-Plex® suspension array system is a flow-based dual-laser system for
simultaneously identifying and quantitating up to 100 different analytes in a
single biomolecular assay (xMAP technology). The system detects and
measures molecules bound to the surfaces of fluorescent microspheres,
providing highly accurate, real-time digital analysis of serum or culture media
samples as small as 50 μl. This allows quantitative analysis of a wide variety of
cell biology assays, including immunoassays, complex genetic assays, and
enzymatic assays.
The Bio-Plex suspension array system consists of a fully integrated array
reader and microplate platform, with an optional HTF (high-throughput
fluidics) sheath delivery module.
Figure 1. Bio-Plex array reader and microplate platform, with optional HTF
1
Bio-Plex Manager Software 6.1 User Guide | Components
The system also includes validation and calibration reagents, a selection of
cytokine and phosphoprotein assays, sample preparation reagents, and a
computer running Bio-Plex Manager
phase, diabetes, cancer and isotyping assays have been introduced with this
version of the software.
Check www.bio-rad.com/bio-plex/x-plex for newly-available assays and
reagents.
The assays contain sets of microscopic color-coded beads, each of which is
conjugated with a different reactant.
Reactants can include:
•DNA
• Enzyme substrates
• Receptors
•Antigens
• Antibodies
These can be used to create, for example, a capture sandwich immunoassay.
To perform a multiplex reading, samples are mixed with conjugated
microsphere and reactant mixtures; next, fluorescent reporter molecules are
added. The assays are loaded into the wells of a 96-well microtiter plate, and
the plate is inserted into the microplate platform. The platform and array
reader are controlled by a computer running Bio-Plex Manager software.
The detection system in the array reader uses two lasers to analyze the
microspheres in a flow stream. The first laser identifies each microsphere and
its associated analyte based on the fluorescent signature of the microsphere,
and the second measures the amount of analyte, using the reporter molecules
attached to the analytes. When the reading is complete, Bio-Plex Manager
displays the raw data and generates detailed summary reports.
™
6.1 software. New angiogenesis, acute
Components
A certified Bio-Rad service engineer will install the complete Bio-Plex
suspension array system at your site, including the array reader, microplate
platform, and computer. The system setup procedure is described in
“Bio-Plex 200 System Hardware Instruction Manual”, available for download
from the Bio-Rad website.
The Bio-Plex suspension array system includes the following components:
• Array reader
• Microplate platform
• Bio-Plex MCV Plate IV (required for Bio-Plex Manager 5.0 and later)
2
Advantages
•Bio-Plex reservoir
• Pentium-class PC, preinstalled with operating system, Microsoft
Excel, Microsoft Internet Explorer, Microsoft.NET, and Bio-Plex
Manager Instrument Control software
• Software package containing Bio-Plex Manager CD-ROM and a
hardware protection key
• Instrument manual for array reader and microplate platform
• Sheath fluid bottle
• Sample needles (two long)
• Protective shield
• Waste bottle
• Communications cable to connect the reader to the computer
• Communications cable from microplate platform to the computer
• Computer monitor
• Power cords
• Computer keyboard
• Computer mouse
• HTF (High Throughput Fluidics), if selected
• Communications cable from the reader to the HTF, if HTF is present
The Bio-Plex suspension array system comes with these reagents:
• Bio-Plex Calibration Kit (CAL1 and CAL2)
• Bio-Plex Validation Kit 4.0 (optics, fluidics, reporter, and classify
components)
• Sheath fluid
Advantages
With the Bio-Plex suspension array system, you can:
• Simultaneously quantitate up to 100 different analytes from culture
media and serum samples as small as 50 μl
• Automatically analyze all the samples in a 96-well microtiter plate—
yielding up to 9,600 data points—in about 30 minutes
• Instantly customize your experiments by mixing Bio-Plex assays or
creating your own assays
• Analyze results, prepare reports, and print and/or export data
immediately after each reading
• Provide a complete electronic audit trail of data generation and
analysis in a secure digital environment with multilevel account
access, that is compliant with the Code of Federal Regulations,
Title 21, Part 11, “Electronic Records; Electronic Signatures”
3
Bio-Plex Manager Software 6.1 User Guide | For More Information
For More Information
For more on the principles and concepts of the Bio-Plex suspension array
system, see Basic Concepts in the Appendix on page 227.
Bio-Rad Technical Support
Bio-Rad Technical Support in the United States is open Monday through
Friday, 5:00 a.m. to 5:00 p.m., Pacific Time. Worldwide technical support is
available on the Web at www.consult.bio-rad.com.
Phone: (800) 424-6723, option 2
Fax: (510) 741-5802
E-mail:LSG.TechServ.US@Bio-Rad.com (U.S.)
LSG.TechServ.Intl@Bio-Rad.com (International)
Web: http://www.consult.bio-rad.com
4
Bio-Plex Manager Software 6.1 User Guide
2 Bio-Plex Manager™
Software Overview
Bio-Plex Manager™ 6.1 software runs on a computer installed with the
Windows XP or Windows 7 operating system, and requires a hardware
protection key, also known as a HASP key, installed on either the computer
itself or the computer network system. The software features a standard
Windows interface, with pulldown menus, toolbars, and keyboard shortcuts.
Bio-Plex Manager comes in two editions—Standard Edition and Security
Edition—and with three available licenses: Instrument Control, Desktop, and
Network, described below. The computer included with the Bio-Plex
suspension array system comes preinstalled with a compatible operating
system and a Bio-Plex Manager Standard Edition, Instrument Control license.
Software Editions
Bio-Plex Manager software comes in two editions:
• Standard Edition gives all users equal access to all features of the
software with no restrictions and no electronic audit trail
• Security Edition provides different levels of user access to various
features and creates a complete electronic audit trail of all data
generation and analysis. The Security Edition can be run in Secure
Mode, with all the security features enabled, or Standard Mode,
which behaves like the Standard Edition of the software
5
Bio-Plex Manager Software 6.1 User Guide | Software Licenses
NOTE: Bio-Plex Manager Security Edition must be installed on the Windows
XP Professional or Windows 7 operating system for full security and
functionality.
Software Licenses
Bio-Plex Manager software is available with three different licenses:
• Instrument Control (previously called Workstation) license enables
the software to control the array reader and microplate platform
and to collect, analyze, and output data
• Desktop license enables the software to analyze data files but not
control the array reader and platform. The instrument
communication and control functions are not available with this
license
• Network license provides Desktop licenses to multiple users over
a computer network. The Desktop license enables the software to
analyze data files but not control the array reader and platform
This user guide assumes that you are using the Standard Edition with an
Instrument Control license, unless otherwise noted. The Security Edition user
levels and restrictions are described in detail in the Appendix on page 219.
Compatibility with Luminex Software
Bio-Plex Manager 6.1 is compatible with Luminex xPONENT software.
Because the Luminex LXR library has been updated, older Luminex IS 2.3
software will not function once you upgrade to Bio-Plex Manager 6.1. You
must upgrade your Luminex IS 2.3 software to the current version in order to
run both applications on the same machine.
If you install Bio-Plex Manager 5.0 or later on a computer with Luminex
xPONENT software, note the following:
• To avoid communication conflicts with the array reader and
platform, do not run Bio-Plex Manager and Luminex software at
the same time.
• If you switch between the two software applications, you must
recalibrate the array reader before acquiring data.
6
ANALYZING LUMINEX DATA IN BIO-PLEX MANAGER
Start up and calibrate array reader
Create a protocol by specifying analytes, plate, and standards
Run the protocol
Review results and generate reports
Print and/or export the data
Bio-Plex Results Generator 3.0 converts CSV Output files from xPONENT 3.1
and 4.0 into Bio-Plex Manager data files. You can analyze the converted
results on any computer running Bio-Plex Manager 4.0 or later. You can make
the files available to an entire workgroup by placing them on a public server.
With earlier versions of Bio-Plex Manager, you had to separately install
Bio-Plex Results Generator. Beginning with Bio-Plex Manager 6.1, Bio-Plex
Results Generator is automatically included in the Bio-Plex Manager
installation.
The Bio-Plex Results Generator 3.0 application (RBXGenerator_3.exe) is
included as a separate product on the Bio-Plex Manager 6.1 software CD.
This allows you to install it on a machine that does not have a Bio-Plex
Manager installation. There is also a help file (Bio-Plex Results Generator 3.0
Online Help) to walk you through installing the Results Generator and setting
up Luminex protocols for conversion.
General Workflow
To collect, analyze, and output data using Bio-Plex Manager software, follow
the general steps outlined below.
.
General Workflow
Figure 2. Bio-Plex Manager software workflow
7
Bio-Plex Manager Software 6.1 User Guide | General Workflow
Quick Guide
The Quick Guide, which displays automatically at
startup, is designed to guide you through the typical
Bio-Plex Manager workflow, from startup and
calibration through shut down.
Commands, such as Start up & Calibrate, open dialog
boxes that contain choices to guide you through the
procedures.
Figure 3. Quick Guide
8
Types of Files
There are two main types of files created and used by Bio-Plex Manager:
• Protocol files (see page 53) contain the settings and controls for
reading a microplate
• Results files (see page 143) contain the data from each reading,
and tools and reports for analyzing that data
The application database file (bioplexdata.mdb) is installed on your hard drive
or file server when you install the software. This database contains calibration,
validation, and instrument operation information. See page 13 for more detail.
Key Software Features
Bio-Plex Manager 6.1 is a single integrated software package that has:
Comprehensive instrument functions
• Data collection
• Maintenance
• Validation
• Automatic error detection and logging
Comprehensive data display
• Raw data presentation including tables, bead maps and histograms
• Customizable tabular presentations
• Customizable standard curve and bar graph displays
Comprehensive Calculations
• Data normalization (internal controls for gene expression analysis)
• Auto calculation and display of usable assay range
• Multiple standard curve fitting options, including Brendan
Scientific, StatLIA, Logistic 5PL.
Comprehensive and versatile automated export options
• Excel (now including optional standard curve graphs)
• Comprehensive .xml file
• New Luminex CSV (comma-separated values) output
• New customizable reports through style sheets
• New custom report for export to custom macros
CFR 21 Part 11 compliance
• User access management
• Comprehensive audit trail and instrument logs
• Secure documents (results files and protocols)
Types of Files
9
Bio-Plex Manager Software 6.1 User Guide | Key Software Features
10
Bio-Plex Manager Software 6.1 User Guide
3 Software Installation
Install, or reinstall, Bio-Plex Manager™ 6.1 software as described in the
following section. If you need to upgrade your software from a previous
version, see the “Bio-Plex Manager 6.1 Software Upgrade and Configuration
Guide” to determine the part number of your upgrade kit.
System Requirements
Component Minimum Recommended
Operating system Windows XP (XP Professional required
for running Security Edition) or
Windows 7 (32-bit)
Processor Pentium 4 or equivalent, 2.8 GHz Core 2, 2.6 GHz or higher
Hard disk space 80 GB 160 GB
System memory 1 GB 2 GB
Screen resolution 1024 x 768 (Windows XP)
1280 x 1024 (Windows 7)
Screen colors 256 colors 24-bit True Color
Ports for connecting
instrument (required for
Instrument Control
license only)
Port for connecting
the HASP key
Other software Internet Explorer 6.0 or later
NOTE: For Windows 7, the Instrument Control version of Bio-Plex Manager 6.1 can be run only on
32-bit; it is not compatible with 64-bit.
1 RS232 serial port and
1 USB port
1 USB port 1 USB 2.0 port
Microsoft Excel 2003 or later
Windows XP Professional
1280 × 1024
1 RS232 serial port and 1 USB 2.0
port
Internet Explorer 8.0
Microsoft Excel 2007
11
Bio-Plex Manager Software 6.1 User Guide | Software Installation
Required Screen Resolution
Your computer screen resolution must be set to at least 1024 x 768 pixels for
correct display of the Bio-Plex Manager interface. The status bar and some
dialog boxes will not display properly at lower resolutions. If your display is
currently set to a lower resolution:
1. Go to the Windows Start menu, select Settings, and select Control
Panel.
2. Open the Display control panel.
3. In the Display Properties dialog, select the Settings tab (see Figure 3).
4. Drag the Screen Area slider to the right (toward More) until you have
selected 1024 x 768 pixels. Click OK to accept the settings.
12
Figure 4. Changing the screen resolution settings
Installing the Software
To install or reinstall the software, insert the Bio-Plex Manager CD into the
CD-ROM drive on your computer.
NOTES
• Before installing Bio-Plex Manager 6.1, we recommend that you
first uninstall any existing version of Bio-Plex Manager on your
computer
• We recommend that you turn off any antivirus protection software
before installation. Such software, if active, can greatly slow the
progress of the installation. If you are unable to turn off your
antivirus protection software, allow 15 minutes for complete
installation. Do not cancel the installation during this period
The Bio-Plex Manager Installation Program opens, displaying a navigation
screen for performing the installation.
Microsoft.NET
Microsoft.NET is automatically installed on your computer when you install
Bio-Plex Manager. It is required to support the logistic curve-fitting features of
Bio-Plex Manager.
Installing the Software
Bioplexdata.mdb File
During installation, you will be prompted to save the application database file
(bioplexdata.mdb) to a location on your hard drive or a file server. This
database file contains logs of calibration, validation, and instrument
operations activity for your instrument.
• For more information about the Calibration Log, see page 34
• For more information about the Validation Log, see page 45
• For more information about the Instrument Operations Log, see
page 51
You can save the bioplexdata.mdb file to any folder on your computer. The
default location is the Bio-Plex Manager application folder. If your computer is
connected to multiple instruments, each instrument must have a separate
bioplexdata.mdb file saved in a different folder.
For more information about the calibration, validation, and instrument
operations logs in Bio-Plex Manager 6.1 Security Edition, see Controlling the
System on page 25.
13
Bio-Plex Manager Software 6.1 User Guide | Software Installation
NOTE: The bioplexdata.mdb file is not compatible with versions of Bio-Plex
Manager earlier than 4.0. If you have an earlier version of the software,
installing 4.0 or later will copy the data from your existing database file
(bioplex.mdb) into the new database. A copy of your old database will remain
in the application folder.
Luminex LXR Directory and Files
A directory called Luminex is automatically created in the Program Files folder
on your computer during installation. Inside this folder is a folder called LXR
that contains various applications for monitoring and communicating with the
array reader and platform. A Windows service called LXService is also
installed and started. This service enables automatic communication with the
instrument when you open Bio-Plex Manager.
Hardware Protection Key (HASP Key)
A hardware protection key, also known as a HASP key, is required to run
Bio-Plex Manager. The HASP key determines the license (Instrument Control,
Desktop, or Network) and the edition (Standard or Security) of your Bio-Plex
Manager. A complete list of HASP key part numbers and the software versions
they enable is in the Bio-Plex Manager 6.1 Software Upgrade and
Configuration Guide.
Instrument Control or Desktop HASP keys must be attached to a USB port on
the computer running the software. Network HASP keys must be attached to
a USB port on the network file server computer.
The HASP key has a driver that is automatically installed when you install BioPlex Manager.
Uninstalling
To uninstall Bio-Plex Manager from your computer, use the Windows Add/
Remove Programs function. Click the Windows Start button, select Settings,
select Control Panel, double-click Add/Remove Programs, and follow the
instructions for removing the program.
14
Bio-Plex Manager Software 6.1 User Guide
4 Starting the System
Before starting Bio-Plex Manager™ 6.1 software, make sure the Hardware
Protection Key is attached to your computer, and switch on the array reader,
the HTF (if one is being used), and the microplate platform. This turns on the
optics inside the array reader and enables communication between the array
reader and software.
NOTE: Avoid using other applications while Bio-Plex Manager is
communicating with the array reader. Running other applications may
interrupt communication between Bio-Plex Manager and the array reader.
Starting Bio-Plex Manager
To start Bio-Plex Manager, click the application icon on your desktop
or select Bio-Plex Manager 6.1 from the Programs directory on your Windows
Start menu. The software opens and attempts to connect to the array reader
and platform.
15
Bio-Plex Manager Software 6.1 User Guide | Starting Bio-Plex Manager
Microplate Platform
Power
Serial port
Array Reader
Power
P1
P2
HTF
Computer
Serial
port
Power
Serial port Power
USB1
USB2
Connecting with the Array Reader and Microplate Platform
The computer running Bio-Plex Manager software is connected to the array
reader by a USB cable, and to the microplate platform by a serial cable. See
the figure below for a diagram of the cable connections.
16
Figure 5. System cable connections
Starting Bio-Plex Manager
Communication between the Array Reader and Microplate Platform
Communication between the computer and the array reader and microplate
platform is automatically established when you open Bio-Plex Manager. If
Bio-Plex Manager is unable to communicate with the instrument, a dialog box
prompts you to check the cable connections.
Figure 6. Connection Error dialog
Make sure the instrument is turned on, check the cable connections, and then
click Reconnect in the dialog. If you are still unable to connect, try restarting
the computer, array reader, and platform. You can also choose the Run
Diagnostic option found under the Instrument menu. This produces a report
which can help Bio-Rad Technical Support determine the cause of the
problem.
Figure 7. Run Diagnostics Report
17
Bio-Plex Manager Software 6.1 User Guide | Starting Bio-Plex Manager
If you do not want to automatically connect to the array reader and platform
when you open the software, select the Do not connect at startup checkbox.
This is useful if you are not connected to an instrument or want to use the
software without connecting to the instrument. To re-enable automatic
connection at startup, select Reconnect from the Instrument menu (see next
section).
Disconnecting and Reconnecting
Communication between the computer and the array reader and platform is
closed when you exit Bio-Plex Manager. If you need to disconnect from the
array reader and platform without shutting down Bio-Plex Manager software
(for example, to troubleshoot the instrument), go to the Instrument menu and
select Disconnect.
To reconnect to the array reader and platform while Bio-Plex Manager is
running, select Reconnect from the Instrument menu. This dialog also enables
automatic connection each time you start the software, if it has been disabled
(see previous section).
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